LIBRARY UNIVERSITY OF CALIFORNIA, UBRARY G Class OUTLINES OF PHYSIOLOGICAL CHEMISTRY OUTLINES OF PHYSIOLOGICAL CHEMISTRY BY s. P.|BEEBE, PH.D. Physiological Chemist to the Huntington Fund for Cancer Research AND B. H. BUXTON, M.D. Professor of Experimental Pathology, Cornell Medical College Neto otfc THE MACMILLAN COMPANY LONDON: MACMILLAN & CO., LTD. 1904 T BIOLOGY LIBRARY G BENEBAL COPYRIGHT 1904 BY THE MACMILLAN COMPANY PRESS or ?HI NEW ERA PRINTING COMPAWV. LANCASTER, PA. PREFACE. THE subject of physiological chemistry is becoming of greater importance every day, but so far as we are aware there is no book of small compass which attempts to deal with the theoret- ical side of the many questions involved. There are plenty of laboratory guides but in order to grasp the significance of his laboratory work the student has to pick up most of his ideas from books which deal primarily with subjects of purely chemical in- terest, and only secondarily with those of special importance to the physiologist. We have endeavored to go straight to the point, dealing only with questions bearing directly on physiological problems. In order to keep the book within reasonable bounds it has been found necessary to assume for the reader some knowledge of inorganic chemistry, and for the same reason it is impossible to avoid making some general statements which would have to be qualified or modified to some extent if taken up in detail. It is sincerely hoped, however, that no actual errors have been committed. Since this is not intended for a laboratory guide, sufficient details for applying various tests have not been given in most cases. The object has simply been to explain the nature of the reactions. LOOMIS LABORATORY, NEW YORK, April, 1904. CONTENTS. CHAPTER I. PAGE. THEORY OF SOLUTIONS. IONIZATION. . . . 1 CHAPTER II. ORGANIC CHEMISTRY OR THE CHEMISTRY OF CARBON COM- POUNDS 22 CHAPTER HI. COMBINATIONS OF THE OXIDATION PRODUCTS OF THE PAR- AFFINS WITH EACH OTHER 36 CHAPTER IV. HALOGEN AND NITROGEN DERIVATIVES OF CARBON COM- POUNDS 54 CHAPTER V. CYCLIC COMPOUNDS 73 CHAPTER VI. THE PROTEIDS 99 CHAPTER VH. ENZYMES . 160 CHAPTER VIH. DISEASE AND IMMUNITY ... -175 Vll OUTLINES OF PHYSIOLOGICAL CHEMISTRY. CHAPTER I. THEORY OF SOLUTIONS. IONIZATION. THE researches of the last fifteen years have made plainer the mechanism of reactions taking place in solution. Practi- cally all of the reactions that are of interest to the physiological chemist take place in solution, so that it is important to know something about the new chemistry. DISSOCIATION. When acids, bases and salts are dissolved in water they are said to dissociate, i. e. y the molecules to a certain extent break apart. These fragments carry a charge of electricity and are called ions. Thus in a 10 per cent, solution of sodium chloride (NaCl) we have H 2 O + NaCl + Na+ -f C1-. Some of the salt exists in the molecular (Na Cl) condition, the remainder being ionized. The metal carries the positive charge and is called the cation (icara, down) ; the acid (Cl) carries the negative charge and is called the anion (ava, up). In an ordinary solution the electrically charged cations and anions are equally distributed throughout, but on passing an electric current through the solution, the cations fly to the cathode or negative ~ pole, and the anions to the anode or posi- tive + pole. Diagrammatically : -f Pole Pole 2 OUTLINES OF PHYSIOLOGICAL CHEMISTRY. Acids, bases and salts dissociate and are called electrolytes. Other substances do not dissociate non-electrolytes. Water (H 2 O) does not dissociate and of itself is a non-conductor. An electric current passed through water is carried solely by the ions in solution. The Na + ion must not be confused with the Na atom. The ion is the atom plus the charge of electricity which causes it to behave very differently. The ions are continually moving about in the solution and whenever a Na + ion comes in contact with a Cl~ ion they com- bine to form for an instant a molecule of undissociated sodium chloride (Na Cl), which, however, immediately dissociates again. It is obvious that the more concentrated the solution the oftener do the ions come in contact and consequently the smaller is the percentage of dissociation. A very dilute solu- tion of most acids, bases or salts is completely dissociated. The dissociation may be measured by : 1. Measuring the elevation of the boiling point of water caused by dissolving a known amount of salt in a known amount of water. 2. Measuring the depression of the freezing point under the same conditions. 3. Measuring the electrical conductivity of the solution. Explanations. 1 and 2. If one molecule of any substance is dissolved in one hundred molecules of any liquid of a dif- ferent nature the lowering of the freezing point or the raising of the boiling point of this liquid is always the same or very nearly so. When acids, bases or salts are dissolved in water the depression of the freezing point is much greater than it should be according to the above law of Raoult. With dilute hydrochloric acid the depression is nearly twice the normal ; with barium chloride three times the normal. THEOEY OF SOLUTIONS. 3 Hydrochloric acid gives two ions and barium chloride three, the ion lowering the freezing point to the same extent as a molecule. For the same reason the boiling point is raised. 3. Water is not dissociable and the conductivity depends on the degree of ionization. Reactions in solutions are reactions between ions. Old method Ha + NaOH = H 2 O + NaCl New method leaving Na and Cl ions in solution. The reaction takes place because when the hydrogen (H + ) ions come in contact with the hydroxyl (OH~) ions, water (H 2 O) is formed which is not dis- sociated, and so the ions (OH~ and H + ) are removed from the sphere of action. The neutralization of any acid by any base is due to the same cause. AgNO s + NaCl = AgCl + NaNO 8 or Na+ +[^+Ag1-fNO; > AgCl. There is reaction because AgCl is insoluble in water and as fast as Ag and Cl ions come in contact they are removed from solution. In potassium chlorate, KC1O 3 , there is a large amount of chlorine but there is no Cl~ ion. K + + C1O~ is the condition in solution. Chloroform, CHCl^ contains much more chlorine than NaCl, but chloroform does not dissociate and consequently it does not show a reaction with silver nitrate. Another example may be given. 4 OUTLINES OF PHYSIOLOGICAL CHEMISTRY. Sulphuric acid (H 2 SO 4 ) added to a solution of barium chloride (BaCl 2 ) I + 2C1- BaSO 4 . In this case as fast as the barium ions are neutralized by the sulphate ions, insoluble BaSO 4 is formed which does not dis- sociate and consequently the barium and sulphate ions are gradually removed from the solution. DISSOCIATION OF ACIDS AND BASES. 1. Acids yield H + hydrogen ions. 2. Bases yield OH~ hydroxyl ions. 1. Acids are substances which give a hydrogen ion when dissociated. Examples : H+ -f Cl- Hydrochloric acid. H+ + NO- Nitric acid. It was formerly supposed that acid properties were due en- tirely to the presence of oxygen. The word means "acid former." We now know that the cause of acidity is the hy- drogen ion. The strength of an acid depends entirely upon the number of hydrogen ions present in a solution of it, L e., upon the extent to which the acid is dissociated. Taking the sulphur acids : H 2 S Hydrosulphuric. H 2 SO 3 Sulphurous acid. H 2 SO 4 Sulphuric acid. We know very well that the one containing the most oxygen is the strongest acid, i. e., the introduction of oxygen facilitates the dissociation of H from the molecule. THEORY OF BOLUTIONS. 5 On the other hand with the acids of phosphorus H 3 PO 2 Hypophosphorous acid, H 3 PO 3 Phosphorous acid, H 3 PO 4 Phosphoric acid, the introduction of oxygen decreases acidity, i. e., phosphoric acid is less dissociated than hypophosphorous acid. In a majority of cases the introduction of oxygen facilitates dissocia- tion and therefore increases acidity but the rule does not always hold good. NOMENCLATURE OF Acros. The method of naming inorganic acids is intended to show the oxygen relations and was adopted when the acid properties were supposed to depend upon the oxygen content. Example. Acids of Chlorine. Acids containing no oxygen have the prefix hydro. HC1 Hydrochloric. HC1O Hypochlorous. HC1O 2 Chlorous. HC10 3 Chloric. HC1O 4 Perchloric. From these acids the following potassium salts may be formed. KC1 Potassium %oVochloride commonly called chloride. KC1O Potassium fo/pochlorite. KC1O 2 Potassium chlorite. KC1O 3 Potassium chlorate. KC1O 4 Potassium perchlorate. Acids ending in ic form salts ending in ate. Acids ending in ous form salts ending in ite. 6 OUTLINES OF PHYSIOLOGICAL CHEMISTEY. 2. Bases are substances which yield hydroxyl (OH) ions on dissociation. The greater the number of hydroxyl ions the stronger the base. The hydroxides of the alkali metals potassium, sodium, lithium, being very soluble, dissociate readily and are the strongest bases. The hydroxides of the alkaline earths, barium, calcium, strontium, dissociate less readily, so are some- what weaker bases. The hydroxides of the heavy metals are but slightly soluble, so are very much weaker. Some of them, like aluminic hydroxide, dissociate in the presence of a strong acid, yielding hydroxyl ions, whilst in the presence of a strong base they yield hydrogen ions. Thus under different conditions they act either as acids or bases, the action being due to the character of their environment. We may have potassium aluminate or aluminum sulphate. Example of the formation of a salt KOH + HNO 8 = KNO 3 + H 2 O K+ cnr + 3 o + K + The latter forming the molecule KNO 3 on concentration of the solution. In representing by equation the reaction between an acid and a base it is therefore always : K iOH -f H| ON0 2 = K O NO 2 + H 2 O not KO ! H +"HO j NO, = K - O - NO, + H 2 O Potassium nitrate, not nitrate of potash. The above gives a general idea of the mechanism of reactions between inorganic compounds taking place in aqueous solution, and the same applies also to a great extent to organic com- pounds. But there are a large number of reactions which take THEORY OF SOLUTIONS. 7 place without dissociation and it is not necessary to refer all of them to the action of + and ~ ions. Chemical activity does not necessarily depend upon ions. CHEMICAL EQUILIBRIUM. 1. Equilibrium of Reactions. When two substances which react chemically are brought together in the same solution the reaction begins at once with a certain velocity, which depends upon the temperature of the solution and its concentration. As the reaction proceeds its speed gradually decreases until the products of the action have attained a definite concentration. This is a condition of equi- librium and no matter how long the substances are left in the solution there is no further change in the concentration. This is generally illustrated by the action of acetic acid upon alcohol. Alcohol -f acetic acid = acetic ester -4- water. The acid begins to act upon the alcohol at once and as it is gradually used up the amount of ester formed in a unit of time decreases until finally we have a condition where the alcohol and acid do not further decrease, and the ester and water do not increase. This is a condition of equilibrium. There is still some free acid and some free alcohol and these two substances continue to react on one another, yet the amount of each in the solution does not decrease. This is explained by the fact that this reaction, like chemical reactions in general, is a reversible one. Reversion. When acetic ester is added to water it reacts with the latter to form alcohol and acetic acid. A simple way of writing the reaction is : Alcohol -f acetic acid * ^ acetic ester -f water. 8 OUTLINES OF PHYSIOLOGICAL CHEMISTRY. The double arrows indicate that the reaction may proceed in either direction. In the first reaction described, as soon as some ester is formed it begins to react with the water to form alcohol and acetic acid, i. e., to reverse the action. The velocity of the reverse action is slow at first because the concentration of the ester is small, but it increases with the latter until finally the amount of ester formed in a unit of time is equal to the amount decom- posed. The condition of equilibrium is then the condition when the velocities in the two opposite reactions are equal. There is a condition of equilibrium for every reaction, but in some cases the action in one direction is so enormously stronger than the action in the other direction, that the former is able to push the latter over almost to a vanishing point, and conse- quently the reaction may appear to be practically complete. The point of equilibrium may be given diagrammatically for three imaginary different reactions. The length of the arrows indicates the vigor in each direction. 505 a- 5QJ? 75* a E E point of equilibrium. This method of considering chemical reactions is of great value to the physiological chemist, and may be approached from another point of view. 2. Equilibrium of Solutions. Suppose we make what is called a saturated solution of sodium chloride. The conditions are as follows : NaCl Solid. 1 NaCl In solution. 2 Ionized. THEORY OP SOLUTIONS. 9 When a solution is saturated as much solid dissolves as pre- cipitates out in a given time. There is therefore equilibrium between 1 and 2. There is also equilibrium between 2 and 3 when the amount which dissociates equals the amount returning to the undissociated condition. There are here two opposing ac- tions. Some of the molecules of salt are breaking apart into ions, whilst some of the ions are combining to form molecules of salt. If we now pass into this satu- rated solution some gaseous hydro- chloric acid, some of it dissolves and dissociates. We thereby increase the number of Cl ions. HC1 ^ H+ + C1-. Since now the Na+ ions come in contact with Cl~ ions more often than they previously did, the com- bining action will exceed the disso- ciation for a time, and more salt will pass into the undissociated con- dition. But the solution is already saturated with undissociated salt, so that some must precipitate out in solid form and this precipitation will continue until a new plane of equili- brium is established. Suppose we have the conditions represented in the drawing. A solution saturated with both hydrochloric acid and sodium chloride and arranged so that pressure can be produced upon the gaseous HC1 above the solution. The conditions of equi- librium may be thus represented : Na + +cr+cr+H + Gaseous HCl FIG. 1. NaCl Solid undissolved. S XaC1 In solution undissociated. Ions. In solution Gas above undissociated. liquid. 10 OUTLINES OF PHYSIOLOGICAL CHEMISTRY. There is complete equilibrium. 1. As much salt dissolves in a unit of time as precipitates from solution. 2. As much salt dissociates into ions in a unit of time as is formed by recombination of ions. 3. As many HC1 molecules fly off from the surface of the liquid in a unit of time as there are molecules entering the liquid from the gas. We now bring the piston down, thereby increasing the pres- sure on the HC1 gas. This causes more gas to dissolve, which in turn increases the number of H and Cl ions. The increase in Cl ions causes an increased formation of undissociated sodium chloride which in turn causes a separation of a small amount of solid salt from the solution. "We might cause further changes by altering the temperature. There is everywhere a continual striving for equilibrium which causes chemical changes and physical changes as well. The factors concerned in the equilibrium of living matter are so numerous and complex that very slight alteration in the system of reactions which make life possible may cause serious results. Complete universal equilibrium is the end towards which we are moving. Catalysis. In the example given above acetic ester in the presence of water decomposes with the formation of alcohol and acetic acid. The rate of decomposition is slow, but if a small quantity of some other acid is added the reaction goes on much faster. The acid added does not apparently enter into the reaction itself, but hastens it to a remarkable degree. The more concentrated the acid the greater the velocity of the reaction. Strong acids have a greater effect than weak acids and experiments have shown that the speed of the reaction is nearly proportional to the concentration of H ions in the solu- THEORY OF SOLUTIONS. 11 tion. The action of the acid is called catalytic and the acid itself the catalyzer. Catalyzers do not enter into the action themselves, but a very small quantity of a catalyzer is able to cause a large effect upon the speed of reaction. The physiological chemist is chiefly interested in the cata- lytic action of the enzymes or ferments secreted by cells with the object of accelerating reactions which are of use to them- selves. Colloids and Oryst-attoids. Soluble substances are divided into two groups, the basis for the classification being their behavior in regard to diffusion. 1. Crystalloids are those which have relatively small mole- cules and readily diffuse through a membrane such as parch- ment. 2. Colloids possess molecules too large to pass readily through pores of a membrane, so diffuse very slowly or not at all. Proteid molecules for instance. In many cases, however, it is not the actual size of the molecules which prevents diffusion, but the fact that the molecules cling together to form what are called solution aggregates. Colloidal Solutions of Metals. It has been found pos- sible within the last few years to obtain some of the metals, gold, silver, platinum, in the condition of a colloidal solu- tion. If two platinum wires carrying a strong electric current are brought close together below the surface of water an electric arc will be formed. Very fine pieces of platinum are torn off from the cathode (negative pole) and the solution soon has a dark brown color. No pieces of platinum can be seen in the liquid even under the highest powers of the microscope. They 12 OUTLINES OF PHYSIOLOGICAL CHEMISTRY. exist however as solution aggregates, not as individual mole- cules. Such metallic colloidal solutions are catalyzers and have cer- tain similarities to enzymes. 1. A very small quantity of metal in this condition can cause a great change in the speed of the reaction. FIG. 2. 2. Certain poisons which destroy enzymes have an analogous action on these colloidal solutions. AGGREGATION, SUSPENSION, AND PRECIPITATION. The solution aggregates being minute collections of molecules are simply small lumps of the substance. In various ways, by the addition of salts for instance, the aggregates can be increased in size so that several cling together. More and more aggregates attach themselves to the larger lumps until these become visible, at first under the microscope and then to the eye, forming a " suspension." Finally the lumps become so large that they sink and the colloid is "precipitated." There is then no real difference between aggregation, suspension and precipitation. They are only different stages of a process. THEORY OF SOLUTIONS. 13 OXIDATION AND KEDUCTION. 1. Oxidation is normally the addition of oxygen to some substance. Reduction is the taking away of oxygen from some substance. Oxidizing agents are substances containing a large amount of oxygen which is easily set free, i. e., it is in loose combination. Reducing agents are substances which have a strong affinity for oxygen and combine with it readily. The above definitions are not sufficient to cover the case. It is a general rule that reduction and oxidation go on simulta- neously but in different substances. CuO + H 2 = Cu + H 2 O. The copper is reduced, the hydrogen oxidized. Copper oxide is the oxidizing agent, hydrogen the reducing agent. It is obvious that oxidation and reduction go on simultane- ously in cases where oxygen is taken from one compound by another. One is oxidized, the other reduced. 2. If the combining power of a metal or electro-positive sub- stances, i. e., substances behaving like metals, is reduced, this is also called reduction. FeCl 3 FeCl 2 Ferric chloride reduced to ferrous chloride. In the first instance the combining power of Fe is with 3 Cl atoms, in the second it only combines with 2 Cl atoms. The reverse of this is called oxidation for convenience although no oxygen is added. FeCl 2 FeCl 3 Ferrous chloride oxidized to ferric chloride. 3. If an electro-negative radical is taken away from an electro-positive radical the latter is reduced. 14 OUTLINES OF PHYSIOLOGICAL CHEMISTRY. Cobalt (Co" 1 ") is a positive radical, being a metal. Chlorine (Cl~) is a negative radical. /; Cl-fH i - >2HC1 C \[C1 HJ The cobalt is reduced, the negative radical being removed. The reverse of this is also called oxidation for convenience The cobalt is oxidized to CoCl 2 . In the first case the hydrogen is oxidized to HC1. Hydrogen is a reducer. At the moment of being freed from combination it is said to be nascent and is then a much more active reducer, i. 6., combines more readily, than when in the gaseous condition. In the gaseous condition it exists as a molecule and may be represented by the formula H H or H 2 whilst nascent hydrogen is in the atomic form H~. We may pass gaseous hydrogen through a solution of ferric chloride for a long time without reducing it to the ferrous state, but on adding acid and then some zinc to the solution, (H 2 SO 4 + Zn == ZnSO 4 + H + H), hydrogen is set free in the nascent condition and the ferric chloride quickly reduced : FeCl 3 + H = FeCl 2 + HCL A very ready means for obtaining nascent hydrogen in aqueous solutions is by the use of sodium or sodium amalgam. Na -f H 2 O = NaOH + H. Oxygen. The oxygen of the atmosphere is in the molecular condition O = O or O 2 . Nascent oxygen may be obtained by the decomposition of hydrogen peroxide, H 2 O 2 = H 2 O -f O, or by the decomposi- tion of water by chlorine, H 2 O -f C1 2 = 2HC1 + O. THEORY OF SOLUTIONS. 15 Ozone is an allotropic form of oxygen represented by the formula A It decomposes very readily, yielding nascent oxygen and is therefore a very active oxidizing agent. AUotropism is a term used to denote the existence of the same element in different physical forms. These forms are probably due to a different atomic arrangement in the mole- cule. Carbon, sulphur, phosphorus and oxygen are prominent elements in living matter and have allotropic forms. OSMOTIC PRESSURE. Matter in the gaseous form is in much the same condition as when in a solu- tion. In both cases the molecules are free to move about, and in both cases the movement expresses itself as a pressure. The pressure exerted by a gas is familiar to all. The analogous pres- sure of the substance in solution, although more dif- ficult to measure, obeys the same laws. It is called the osmotic pressure. This was first demonstra- ted by filling a bladder with alcohol and then immersing it in water. The water can pass in, but the alcohol cannot pass out A Porous cup contaln- ,ing sugar solution B Jar of water C Pressure tube FIG. 3. 16 OUTLINES OF PHYSIOLOGICAL CHEMISTRY. to any extent, and the increased pressure will cause the bladder to burst. The demonstration of osmotic pressure requires a semi- permeable membrane, i. e., a membrane permeable to the sol- vent, but not to the dissolved substance. Such a membrane may be prepared by precipitating copper ferrocyanide in an unglazed earthenware cup. If such a cup is filled with a sugar solution, attached to a manometer and then immersed in a jar of water the manometer will soon indicate a rise of pressure in the cup. This pressure will continue to in- crease until the final pressure will be the same as it would be if the dissolved substance were a gas and occupied the same volume as the sugar solution. The pressure attained depends upon the concentration and temperature of the solution. The explanation of gas pressure in terms of the kinetic theory is that it is due to a bombard- ment of the walls of the confining vessel with particles of gas but there is no satisfactory explanation of the cause of osmotic pressure. The many problems of filtration, secretion and absorption in the body have caused physiologists to be very much interested in the phenomena of osmotic pressure. CALCULATIONS OF A FORMULA. To calculate the formula of a compound two things must be known : 1. The molecular weight. 2. The percentage composition. The molecular weight may be determined by : 1. Measuring the elevation in boiling point or depression in freezing point caused by dissolving a known amount of the compound in a known amount of liquid. THEORY OF SOLUTIONS. ' " - 2. By determining its vapor density, provioitrg--it i wifl vola- tilize without decomposition. 3. Other methods which cannot be described. The percentage composition is determined by ultimate analy- sis of a pure sample of the compound. Suppose we have a compound with a molecular weight 180, percentage composition C 40 per cent. H - 6.66 O - 53.3 " The percentage of each element is divided by the atomic weight of that element. C - 40/12 - 3.33 1 H- 6.66/1 -6.66 2 O - 533/16 - 3.33 1 Express the ratio in whole numbers and the simplest formula for the given substance would be CH 2 O which would have a molecular weight of 30. The compound in question had a molecular weight of 180 so it must be six times as large as the simpler one or C 6 H 12 O 6 . The difficulty of determining the molecular weight of many substances prevents an empirical formula from being exactly determined. Two substances may have the same percentage composition and yet have a very different molecular weight, as in the example given above, formaldehyde and dextrose. The empirical formula of a compound shows the number of atoms of different elements there may be in a molecule but it does not show their arrangement. The behavior of any com- pound is much more intelligible if we know how the different atoms are combined. This atomic arrangement is shown by the graphic formula : 2 18 OUTLINES OF PHYSIOLOGICAL CHEMISTRY. H 2 SO 4 empirical formula. (1) g^ graphic formula. There are obviously other ways of arranging the atoms in this case, X Q H (2) \(] or (3) H 7 Sulphur may have a valence of 4 or 6, oxygen always being 2, hydrogen 1. There are many reasons why formula 1 is to be regarded as correct. In the first place compounds having oxygen combined with itself are very unstable H 2 2 , H-O-O-H, or O 3 ozone / \ Sulphuric acid is very stable, therefore it cannot be either (2) or (3). For the same reasons HNO 3 is written ^ and HC1O,,, /> _tL O v- '1\. \> Cl when oxidized may have 1, 3, or 5 valences. THEORY OF SOLUTIONS. 19 There are reasons also for believing that the hydrogen is tied to the sulphur by means of oxygen. SO,, combines with chlorine to form a compound this reacts with water to form sulphuric acid. H O ("H ajx^ H-o-[H.aj/ REASONS WHY REACTIONS TAKE PLACE. An old rule which applies to a large number of cases is the following : " Whenever an insoluble or volatile substance can be formed, it will be formed to the full extent of the compo- nents." As an example we may consider the decomposition of calcium carbonate by heat : CaCO 3 ^"^ CaO + CO 2 . CO 2 is volatile and as long as there is no hindrance to its escape the decomposition continues. If, however, the calcium carbo- nate is heated in a strong closed steel cylinder so that the CO 2 cannot escape, the calcium carbonate is found unchanged when the cylinder is opened after cooling. The reactions which are used in ultimate analysis obey this law. In determining the amount of sulphur in organic sub- stances the sulphur is oxidized to a sulphate which is precipi- tated by some soluble salt of barium, such as the chloride. , + BaCl 2 2 Nad + BaS0 4 20 OUTLINES OF PHYSIOLOGICAL CHEMISTRY. The BaSO 4 is almost completely insoluble and is therefore removed from the solution. By adding an excess of barium all the sulphate will be precipitated. The reaction will run to an end practically. This is a case where the equilibrium is at vanishing point. A few graphic formulas of inorganic compounds which will be of use are given. They will serve also as an introduction to the numerous graphic formulas of organic compounds with which we shall have to deal. Fe, 2 or 4 valences, Cl, 1 valence. Cl Cl a Fe^-Cl \C1 Ferric chloride, Fe 2 Cl 6 , usually written FeCl 3 . Ferrous chloride, FeCl 2 . Basis for ferri compounds. Basis for ferro compounds. S, 2, 4 or 6 valences, H, 1 valence, O, 2 valences. ,H O O H-O-S-O-H H-O-S-O-H II O Hydrosulphuric acid. Sulphurous acid. Sulphuric acid. Ba, 2 valences, Ca, 2 valences. /O H Ba< Ba<^ Ba< /Cl < \C1 Barium hydroxide. Barium chloride. Barium sulphate, Calcium (Ca) combines in the same way. K y 1 valence, Na, 1 valence. O O H-0-S-O-K O Monopotassic sulphate, KHSO 4 K-O-S-O-K O Dipotassic sulphate, K 2 S0 4 Na combines in the same way. P, 3 or 5 valences. THEORY OF SOLUTIONS. 21 H N O H H O N = O H O Ammonia gas, Ammonium hydroxide, Nitrous acid, Nitric acid, NH 8 NH,iOH(OH- ion) HNO 2 (H+ion) HNO 3 (H+ion). ULTIMATE ANALYSIS. The determination of the elementary content of any substance is called ultimate analysis. The elements which occur in proteid and are therefore of most interest to us are C, O, H, P, S and N. The first three of these are estimated in various ways, but hardly concern us since it is only exceptionally that determina- tions of C, O and H content of proteid are made. P, S and N estimations however are constantly required. P Content. The P in the substance is oxidized to phos- phoric acid and precipitated as insoluble magnesium phosphate and the amount of P calculated. In the words of the chemist it is estimated as P 2 O 5 . 8 Content. The sulphur is completely oxidized to sulphuric acid or soluble sulphates, then precipitated as the insoluble BaSO 4 , which is weighed and the amount of S calculated. Estimated as barium sulphate. N Content. N is estimated as NH 3 by KjeldahPs method. (see text-books). CHAPTER II. ORGANIC CHEMISTRY OR THE CHEMISTRY OF CARBON COMPOUNDS. THE element carbon differs from other elements in its re- markable power of combining with itself to form stable com- pounds. Its combining power or valence is determined by an analysis of many of its simplest compounds to be 4, i. e., it .can combine with four atoms of hydrogen. If we consider the structure of the compounds of hydrogen and oxygen, H 2 O and H 2 O 2 we see a reason for the difference in properties due to the fact that oxygen is combined with itself. H O H is one of the most stable compounds known. H O O H is very readily decomposed, the element of weakness being the tie between the O atoms. Again the diazo compounds which contain the atom group N = N are very explosive, i. 6., the compound is unstable, the two N" atoms being easily separated. But carbon may com- bine with itself (C C C C) to an almost unlimited extent, and still form stable compounds. It is this property of the C atom which makes possible the immense and complicated molecules of organic matter. The simplest compounds of carbon are the hydrocarbons, substances which contain only carbon and hydrogen. The simplest of the hydrocarbons is represented by the formula CH 4 . Because of the large number of hydrocarbons and their great variation in structure it is necessary to arrange the atoms graphically in order to grasp the full significance of their con- struction. The graphic formula of CH 4 may be thus written 22 ORGANIC CHEMISTRY. 23 H C H H Methane (marsh gaa). Methane is the simplest of a series of compounds called the methane or marsh gas series of hydrpcarbons. The H atoms may be replaced by other elements, forming what are called substitution products. For instance if chlorine is mixed with methane in equal volume and exposed to sun- light, a reaction which may be represented as follows, takes place. H H 01 H H ">c/ | >HCI+ \c/ H in cij H ci Methane + chlorine = hydrochloric acid + monochlonnethane. CH, + Cl a = HC1 + CH 3 C1 On increasing the amount of chlorine the change may go further H H Cl H H X-JL >HC1+ >\ ci IL.CIJ ci ci Monochlormethane. Dichlormethane. CH 3 C1 + C1 2 = HQ + CHjCl, By continued action a series of chlorine substitution products of methane are formed. CH 3 C1 Monochlormethane, CH 2 C1 2 Dichlormethane, CHC1 3 Trichlormethane, CC1 4 Tetrachlormethane. In the same way iodine and bromine substitution products may be formed. 24 OUTLINES OF PHYSIOLOGICAL CHEMISTRY. These compounds are said to be saturated because the maxi- mum combining power of the C atom is used. The next in this series of hydrocarbons contains two atoms of carbon held together by a single bond, the three remaining valences of each C atom being brought to saturation by hydro- gen. The only possible way of representing this graphically is by the formula H C H H C H H Ethane, C a H e . Ethane is a gas having much the same chemical behavior as methane. We may consider it as a substitution product of methane, formed by substituting a methyl group, CH 3 , for one of its H atoms. In the same way a methyl group if substituted in ethane, gives propane, the next higher compound of the series. Propane, C 3 H 8 or CH 3 .CH a .CH, . It is evident from the graphic formula of propane that the central C group differs from the other two, and therefore, on forming the next higher compound of the series it will make a difference whether we substitute the methyl group on one of the C atoms at the end of the chain or the central one. We may thus form two hydrocarbons having the formula C 4 H 10 (butane), but differing in the arrangement of the groups. ORGANIC CHEMISTRY. 25 Normal butane, C 4 H 10 . Iso-butane, C 4 H 10 . The two compounds, although they have precisely the same amount of C and H, present different characteristics reac- tions, appearance, odor, etc. which can only be explained on the basis of differing internal arrangement. Such compounds are said to be isomers and as we go higher in the series the number of possible arrangements and conse- quently the number of isomers increases with alarming rapidity. Methane CH 4 - 1 Pentane C 5 H 12 - 3 Octane C 8 H 18 - 18 Ethane C 2 H 6 - 1 Hexane C 6 H 14 - 5 Nonane C^ - 35 Propane C 3 H 8 1 Heptane C 7 H 16 8 Decane C^H^ 75 Butane C 4 H 10 - 2 A common difference CH 2 exists between the successive members so that a general formula for the series may be given CH 2n+2 . The hydrocarbons are such inactive bodies chemically that they are of no particular interest to us except to take as simple examples with a view to gaining some elementary ideas of the structure of organic compounds. The lower (those with a few C atoms) members of the series are gases, and as the molecular weight increases the boiling point rises ; the higher members being the wax-like bodies called paraffins. Such a series as that given above in which there is a simi- larity in constitution but a gradual and regularly increasing variation in properties is said to be homologous, and the mem- bers are called homologues. 26 OUTLINES OF PHYSIOLOGICAL CHEMISTRY. Example : Butane, C 4 H 10 , is a higher homologue of propane, There are many such series among the organic compounds. By oxidation of the hydrocarbons a number of interesting and well-known compounds, the alcohols, aldehydes and organic acids are obtained. We can now trace in regular sequence the processes of oxi- dation and the compounds resulting from it. ALCOHOLS, ALDEHYDES, ACIDS. 1 . Alcohols. If methane is completely oxidized the result is the formation of carbon dioxide and water. If the oxidation is carefully regulated however the inter- mediate products in the process may be obtained. We may thus introduce one O atom into the methane molecule = H C H In this new substance the H atom which is tied to the carbon through the oxygen atom behaves very differently from the remaining three, being much more easily split off, thus allow- ing the O to attach itself to other compounds. In other words the new compound is chemically active whilst the hydrocarbons are chemically inert. The substance CH 3 OH is the simplest of a new homologous series of compounds obtained by partially oxidizing the hydro- carbons. These are the alcohols, the one already given being methanol (methyl alcohol), commonly called wood alcohol be- cause it is obtained by the dry distillation of wood. ORGANIC CHEMISTRY. 27 The alcohols are always designated by the suffix " ol." H C O H H-C H H C H H C O H H-C H H C H Butanol and higher homologues. A H H Methanol (methyl alcohol), CH 3 OH. Ethanol (ethyl alcohol), Propanol (propyl alcohol), C 3 H T OH. General formula C n H 2n+1 OH. The alcohol of everyday use is ethanol or ethyl alcohol obtained as a product of fermentation. The alcohols exist in isomeric forms but since two or more isomeric alcohols may be derived from one hydrocarbon the number of isomers is vastly greater. Fortunately only a few are of importance for us. Alcohols are divided into three classes, primary, secondary and tertiary, the basis of the classification being the arrange- ment of the molecule. This may be illustrated by the butyl alcohols C 4 H 9 OH. QH, CH 2 OH Normal butane, C 4 H 10 . Primary normal butanol (primary butyl alcohol), CH S Secondary normal butanol (secondary butyl alcohol), C 4 H 9 OH. COH CH, Iso butane, C 4 H 10 . Primary iso butanol (primary Tertiary butanol (tertiary butyl-alcohol), C 4 H 9 OH. ' butyl-alcohol), C 4 H 8 OH. 28 OUTLINES OF PHYSIOLOGICAL CHEMISTEY. GROUPS CHARACTERISTIC OF ALCOHOLS. (1) E CH 2 OH Primary (2) ^CHOH E/ Secondary. (3) X E-)COH W Tertiary. 2. Aldehydes, Ketones. The alcohols have different char- acteristics, but the differences in their oxidation products is more striking. A. Primary alcohols on further oxidation yield aldehydes. E c O H t But since the C atom can never support more than one chemically active (OH) group, this combination is an impossi- bility, so something must happen. H 2 O is immediately thrown off and the C attaches itself to the remaining O by two valences. E-C-0 iH = B-e=:0+] A ! Aldehyde. 1 The group CHO is characteristic of aldehydes. B. Secondary alcohols, on oxidation yield ketones. ORGANIC CHEMISTRY. 29 Again H 2 O is thrown off and we get Ketone. The group CO is characteristic of ketones. Since the tertiary alcohols have the formula E COH there is obviously no H left for oxidation. If the COH group is further oxidized to C = O, the C atom must take a valence from one of the radicals, so that the compound would break up. The aldehydes are always designated by the suffix "al." Thus we have : H C=O H C=O H C=O iY ATT Atr Butanal and ^^3 V n 2 higher homologues. ATT LJ1 3 Methanal Ethanal Propanal - ,, (progtaic.Meh.de), Formalin is the commercial formaldehyde 40 per cent, in water. The Ketones obviously cannot exist unless the chain contains three or more C atoms. They always have the suffix " one." Pentanone and higher homologues. H 3 Propanone (acetone), Butanone, C a H 6 . CO. C 3 H 8 . CO. i C H 30 OUTLINES OF PHYSIOLOGICAL CHEMISTRY. 3. Acids. The aldehydes K- have one H left which can be oxidized to form an acid. K C-H -fO K C O-H Aldehyde. Acid. Acids always have the suffix acid so we have O=Q O H Bu1 homologues. Butanacid (butyric O== C-O-H O=C-O-H fcn, "ffiSSC* H CH 3 CH 3 Methanacid (formic Ethanacid (acetic Propanacid (propionic acid), HCOOH. acid), CH 3 . COOH. acid), C 3 H 6 . COOH. General formula C n H 2n O 2 . The group COOH is characteristic of the acids. The Ketones have no H left for oxidation. On further oxidation the com- pound breaks up. Summarizing On oxidation. Primary alcohols -> Aldehydes m-*- Acids ->- Break up. Secondary alcohols -- Ketones -> Break up. Tertiary alcohols a->- Break up. Acids then can only be derived from primary alcohols, (CH 2 OH) the series so formed being called the Fatty acid series, since certain of the higher homologues encer into the composi- tion of the fats. The members of the fatty acids series are monobasic, since there is only one OH group to enter into combination. ORGANIC CHEMISTRY. A comparison may be made with the mineral acids. K c O H O 31 H-0-S-O-H Fatty acid monobasic. Nitric acid monobasic. Sulphuric acid dibasic. The following table summarizes the four homologous series so far discussed. Chemically Inert. Chemically Active. Basic. Neutral. Acid. General Prefix. Hydro- carbons. Alcohols. Suffix "ol." Aldehydes. Suffix '' al." Acids. Suffix "acid." Meth. Eth. Prop. But. CH< !: C^HIO CH 8 OH C 2 H 5 OH CsH 7 OH C 4 H 9 OH H.CHO CH 3 .CHO CjHoCHO CaHj.CHO H.COOH CH,.COOH C 3 H 6 .COOH C S H 7 .COOH and so on for the higher homologues, which are designated by the number of C atoms they contain Pent., Hex., Hept., etc. A study of the table brings out some interesting points. 1. The introduction of O changes chemically inert into chemically active compounds. This rule is not universal but holds good for many organic compounds. 2. The introduction of a little O produces basic alcohols which will readily unite with acids : Example : H a C-:0-H Hi f - O CH, JLC-O-NO, CH. O=N=O Ethyl alcohol + Nitric acid = Ethyl nitrate + water. +H,0 The alcohols of this series have only one active OH group, so are called monoatomic. 32 OUTLINES OF PHYSIOLOGICAL CHEMISTEY. 3. The introduction of a little more O produces neutral alde- hydes, which have no special affinity for either acids or bases, but being chemically active afford some very interesting reac- tions with various organic compounds. These reactions will have to be discussed later since they occur with complex substances, the constitution of which has not yet been considered. The reaction with Fehling's solution, h'owever, will be explained directly. 4. The introduction of a further amount of O produces acids which will readily combine with a base. Example : A _ K Acetic acid + Potassium hydroxide = Potassium acetate + water CH, COOH KOH CH 3 . COOK It has been explained in Chapter I. that an increase of O does not necessarily mean an increase of acidity, but with organic compounds this is nearly always the case. It may however be remarked that the alcohols are weak bases and the fatty acids weak acids. 1. An alcohol in combination with an acid is easily driven out by a strong mineral base such as KOH. Example : CH 3 CH 2 O NO 2 + KOH = K O NO 2 + CH 3 - CH 2 OH Ethyl nitrate + Potassium hydroxide = Potassium nitrate + Ethyl alcohol. 2. A fatty acid in combination with a base is easily driven off by a strong mineral acid. Example : CH 3 COOK -f HONO 2 = KONO 2 + CH 3 . COOH Potassium acetate -j- Nitric acid = Potassium nitrate + Acetic acid. ORGANIC CHEMISTRY. 33 TESTS. 1. For Alcohols. lodoform test, see p. 54. 2. For Aldehydes. The aldehydes representing an inter- mediate stage of oxidation are always eager for more O, and take it up whenever it is offered in an accessible form. They will therefore seize O from other compounds in cases where the latter carry it loosely. Such a compound is found in cupric hydroxide. Aldehydes therefore are reducers. In cases where it is required to deprive certain compounds of their oxygen, or a part of it, the aldehydes are often employed for this purpose. CHEMISTRY OF FEELING'S SOLUTION. Copper has two hydroxides and corresponding insoluble oxides. Cu O H Cu v ,O H >O Cu< Cu=O Cu O H Cu/ \O H Cuprous hydroxide Cuprous oxide Cupric hydroxide Cupric oxide yellow. red. blue. black. Cupric hydroxide, C^OH)^ is blue when first precipitated. On adding alkali (KOH) and boiling, H 2 O is driven off and black cupric oxide CuO which is insoluble is formed and falls as a precipitate. Cu(OH) 2 = CuO + H 2 0. But if Cu(OH) 2 is heated with a reducing agent such as an aldehyde (glucose is an aldehyde), the cupric hydroxide decom- poses still further to cuprous oxide. 2Cu(OH) 2 = Cu 2 O + H 2 O + O, the O being taken up by the reducer. The bright red Cu 2 O is insoluble and this is the red precipi- tate which is looked for on testing solutions for the presence of reducers glucose, etc. 3 34 OUTLINES OF PHYSIOLOGICAL CHEMISTRY. But unless there is enough reducer to change all the cupric hydroxide into red cuprous oxide, some of it will precipitate as black cupric oxide which masks the red color. To avoid this tartaric acid is used, which forms a soluble colorless compound with the cupric oxide but does not affect the red cuprous oxide. The point is to add something by which the CuO is made soluble and colorless, but which leaves the Cu 2 O unchanged. Fehling's solution A is CuSO 4 . Fehling's solution B is KOH -f some tartrate. On mixing the two the following reaction occurs : CuS0 4 + 2KOH = K 2 S0 4 + Cu(OH) 2 The Cu(OH) 2 must be used fresh as it decomposes slowly in the cold, so the two solutions are not mixed until just before using. Nylander's Test. Glucose and other aldehydes reduce other metallic oxides in alkaline solution. The reduction of bismuth oxide is the basis for Nylander's test. In this case the metallic oxide in alkaline solution will keep for months. In boiling with a reducing substance a black deposit of bismuth is formed. The Ketones (CO) have no spare H for oxidation so are not greedy for O, and do not reduce copper oxide. The reaction with Fehling's solution is therefore an aldehyde, not a ketone reaction. 3. For Acids. Treated with FeCl 3 the lower (homologues of the) fatty acids and their salts, formates, acetates, etc., afford a red color due to the formation of colored Fe compounds. The reaction is not well understood and need not be discussed in detail. UNSATURATED COMPOUNDS. In addition to the homologous series already discussed there are others called unsaturated compounds, in which two C atoms ORGANIC CHEMISTRY. 35 are joined by two or three links instead of one. The hydro- carbons of such series have the suffix ene and ine. Clia CU CHg CHj OH Ethane. Ethene (ethylene gas). Ethine (acetylene gas). S H I CHa Propine. These likewise form long series of oxidation and substitution products but the compounds formed are of little interest to physiological chemists, who do not deal in gases and mineral oils. It will be noticed that in these compounds the C atom always has four occupied links, but since in the unsaturated hydro- carbons there are two adjacent carbon atoms united doubly or trebly to each other the ratio of hydrogen to carbon is smaller than in the saturated series. THE CARBON MOLECULE. The C atom can attach itself to another or to two others by all of its four valences, thus forming the carbon molecule. Three forms of allotropism are possible which are probably represented by charcoal, graphite and diamond. C i\ /~i /"t /~i /"^ ml XV %/ C/^i fN Vi \j n " \j \j 123 Only one grouping is possible with 1, but it is obvious that the rings 2 and 3 can theoretically contain an unlimited num- ber of carbon atoms, but nothing is known of the way in which the carbon atoms hang together to form the molecule. CHAPTER III. COMBINATIONS OF THE OXIDATION PRODUCTS OF THE PARAFFINS WITH EACH OTHER. ETHERS, ESTERS, ANHYDRIDS. THE ethers are the result of a reaction between the OH groups of two alcohols with loss of water ; in other words of two bases. Strong mineral bases cannot do this, but weak organic bases are able to. Example. Ethanol. CH 3 CH 2 j""H j CH 3 CH 2 + ^>0+H 2 Ethanol. CH,-CH 2 I OH | CH 3 -CH 2 Ethanether (ether). The alcohols which enter into the combination are not neces- sarily similar. The ether may be methyl ethyl, or ethyl propyl, etc. CHj CH 3 > CH 8 -CH 2 CH 3 -CH 2 -CH 2 The ether of the laboratory is ethyl ether, often called sul- phuric ether, although there is no sulphuric acid in its compo- sition. Sulphuric acid, however, is employed in its manufacture from alcohol. The reaction occurs in two stages : CH 3 .CH 2 JOH1 ft I) + | Hi O S O H = CH 3 CEL O S O-H+H-O O > Alcohol + Sulphuric acid. Ethyl sulphuric acifi. 36 OXIDATION PRODUCTS OF THE PARAFFINS. 37 2. The ethyl sulphuric acid reacts with a second alcohol molecule. CH..CH, -o v ^o CH 8 .CH, + H,SO t + CH, CH; H /0 ' ^O CH v >0 8 CH 2 The H 2 O is not liberated but immediately goes to recon- struct the sulphuric acid. This is an example of catalysis where the active agent in a reaction is not itself affected except perhaps temporarily as in this instance (p. 10). Theoretically therefore a given amount of H 2 SO 4 should be able to convert an unlimited amount of alcohol into ether. But owing to secondary reactions the H 2 SO 4 is in practice gradually exhausted. The esters are the result of a reaction between the OH group of an alcohol and the OH group of an acid with loss of water, i. e.j of an organic base with an acid. Example. Ethanoi. CH8 . CHa I OH CH 3 - OT, + )>0 + H 2 Ethan acid. CH 3 COO | H CH 3 CO Ethanacetester. As with the ethers the alcohols and acids are not necessarily corresponding homologues, so that one can have methylacet- ester, ethylpropanacidester, etc. The acid may be mineral as well as organic. CH, CH 2 CH 3 .CH 2 |OH = \0 +H 2 Nitric acid. O 2 N O j H O 2 N Ethyl nitrate. 38 OUTLINES OF PHYSIOLOGICAL CHEMISTRY. The Anhydrids are formed by reaction between the OH groups of two acids with loss of water : Example. Acetic acid. CH S ' COO j H CH 3 ' CO Acetic acid. CH S - CO 1 - OH / + H ' CH 3 - CO Acetanhydrid. The ethers, esters and anhydrids may therefore be shown in diagrammatic form as follows : Alcohol v Alcohol v Acid v Alcohol / Acid / Acid / Ether. Ester. Anhydrid. The gases carbon monoxide C = O and carbon dioxide O=C=O may be regarded as anhydrids, since they may be obtained by driving off water from formic and carbonic acids. r 2 = H-O-C-H O=O-}-H 2 O = H O C H Formic acid. O=C=0+H 2 O = H C-O-H Carbonic acid. It has been said that the C atom cannot support more than one OH group. This is true as a general rule but an exception must be made in the case of carbonic acid. Carbonic acid how- ever only exists as such in solution in water, and is easily decomposed, CO 2 gas being driven off. It is therefore unstable having a tendency to throw off H 2 O on the slightest provoca- tion. Its salts however are stable, e. g., K 2 CO 3 ; CaCO 3 . It has also been said that every C atom must have all four valences occupied. Carbon monoxide is the single exception to this. It is formed on combustion with insufficient supply of oxygen. OXIDATION PRODUCTS OF THE PARAFFINS. 39 DIATOMIC ALCOHOLS. A second group of a chain may become oxidized, so that two OH groups are present instead of one. CH 8 CH 2 OH CH 2 OH CH S CH 3 CH,OH thanol (alcohol), Ethandiol monoatomic. diatoi Ethane. Ethanol (alcohol), Ethandiol (glycol), " >mic. Of these two OH groups one may become further oxidized to an acid, forming an oxyacid, and again both groups may become oxidized to form a diacid. The intermediate aldehydes need not be considered in detail. CH 2 OH COOH COOH CH 2 OH CH 2 OH COOH Ethandiol (glycol). Ethanoxyacid, Ethandiacid, (oxyacetic acid), (oxalic acid), monobasic acid. dibasic acid. In the same way we may have propandiol and diacid, or butandiol and diacid, etc., propanoxyacid and so on. Graphic formulas of these and many others should be constructed in order to fully grasp the situation. TRIATOMIC ALCOHOLS. There, may be three groups oxidized to OH and in this case we have a triatomic alcohol. CH 2 OH CHOH CH 2 OH Propantriol (glycerin or glycerol). In like manner alcohols may be tetratomic, pentatomic, etc. Mannite is an alcohol with six C atoms all of which are oxi- dized to OH. It is therefore a hexatomic alcohol. 40 OUTLINES OP PHYSIOLOGICAL CHEMISTRY. It is obvious that in the case of a diacid the acid radicals must be at either end of the chain since an acid radicle requires three valences = O and OH, whilst two only are available in the middle C atoms, but with the oxyacids the alcohol OH may be attached to any of the C atoms. According to the position of the alcohol OH group with regard to the acid group the oxyacid is called alpha, beta, gamma, etc. yCH 3 yCH 3 yCH 2 OH Butanacid a oxybutanacid oxybutan y oxybutan (butyric acid), (a oxybutyric acid). (butyric) acid. (butyric) acid. Lactic Acid is oxypropanacid and it is evident that the OH group may hold either one of two positions. COOH HCOH CH 3 CH 2 OH oxypropanaci (ethylene lactic acid). a oxypropanacid |8 oxypropanacid (ethylidene lactic acid). ASYMMETRIC CARBON ATOM AND ITS ACTION ON POLARIZED LIGHT. Taking the middle C of the a oxypropanacid we find that there are four different groups of atoms attached to it, COOH, CH 3 , H and OH, whilst the middle C of the ft acid has only three different kinds attached, COOH, CH 2 OH and H. The former is an asymmetric C atom and the latter a sym- metric C atom. Any C atom to which 2, 3, or 4 similar atoms or groups of atoms are attached is symmetric. Any C atom which has four different groups attached to it is asymmetrical. If the molecule is regarded as existing in three planes OXIDATION PRODUCTS OF THE PARAFFINS. 41 (stereo-chemistry), as no doubt is the case in nature, we can construct a diagram of it as follows : 1. Asymmetric. 3. Symmetric. If the attached groups are arranged in a different order as 2. Asymmetric. 4. Symmetric. it will be found that molecule No. 1 cannot be placed directly over molecule No. 2 in such a way that each group lies over a corresponding group. But if the molecules are turned over in opposite directions, the groups will then correspond. One is the mirror image of the other. With a symmetric C atom (3) and (4), no matter how the two or more similar groups may be placed, one molecule can always be superimposed upon another in such a way that each group has a corresponding one underneath it. There is no necessity to turn the molecules over. 42 OUTLINES OF PHYSIOLOGICAL CHEMISTRY. With an asymmetric C atom no matter how the groups are arranged one molecule can always be made to fit on another, provided they are turned over as shown in the diagram. If a small model is made with wire and differently colored balls this fact is made obvious at a glance. With an asymmetric C atom then there are always two dif- ferent combinations possible, but no more. One of these combinations when in solution always has the power of turning the plane of polarized light to the right (dex- trorotatory) and the other to the left (Isevorotatory). Why this is so is not clearly understood ; the fact has to be accepted. When an equal number of such right- and left-handed molecules are in solution together, they neutralize each other and the solution has no power over polarized light. It is the inactive or racemic form of the compound. Alpha lactic acid may exist in three different forms, all of which give similar reactions, except that one is dextrorotatory (d) another Isevorotatory (I) and the third inactive ('), whilst ft lactic acid exists in one form only. The a and /3 lactic acid are chemical isomers. The differ- ences in their make-up are due to different chemical combina- tions. The differences between d and I lactic acid are simply differences of configuration. They are physical or space isomers. Following up the question of the asymmetric carbon atom we may consider a, @ dioxybutyric acid COOH . CHOH . CHOH . CH 3 and constructing its graphic stereochemical formula in three planes, find that it has two asymmetric C atoms. No single one of these can be so placed over another that all the groups correspond, but if 1 and 2 are turned so as to face OXIDATION PRODUCTS OF THE PARAFFINS. 43 each other their groups correspond. 1 is the mirror image of 2, and the same is the case with 3 and 4. There are therefore two right-handed, two left-handed and two racemic forms, or we may say there are four space isomers. )H 1234 Tartaric add is butandioldiacid, COOH . CHOH . CHOH . COOH. So this also has two asymmetric C atoms, but in this case each of the two central C atoms has similar groups attached, COOH, OH and H. Taking 1 and 2 we find that the groups A attached to the upper C atom do not correspond with the groups E attached to the lower C atom. Nor do the groups A form a mirror image of the groups E. But the entire molecule 1 forms a mirror 44 OUTLINES OF PHYSIOLOGICAL CHEMISTRY. image with the entire molecule 2. 1 and 2 are dextro (d) and laevorotatory (I) respectively, and together are inactive or racemic. On studying 3 however we find that the groups A are the mirror image of the groups J5. These two therefore neutralize each other by what is called internal compensation and the molecule in solution is inactive. The same is the case with 4, and since each of these is of itself inactive a mixture of the two is also inactive. 3 and 4, either singly or mixed, form only one single inactive compound, mesotartaric acid. Tartaric acid therefore has three space isomers or four forms instead of six as with /? dioxybutyric acid. The latter is said to be an asymmetric molecule whilst tartaric acid is a symmetric molecule. The dextrorotatory may be separated from the Isevorotatory form in a racemic solution of tartaric acid : 1. By sorting the crystals which are formed with some optically inactive substance. The d crystal has a slightly dif- ferent form from the I crystal ; one being a mirror image of the other. 2. By the different solubilities of certain salts of the two forms. The strychnine salt is commonly used. 3. Certain moulds, e. g., penicillium glaucum, if grown on a racemic solution, feeds on the d form and destroys it, leaving the I form untouched. Fats. Three of the higher monobasic (fatty) acids are of special interest in this connection and may be referred to here : They are ; COOH COOH CH, CH 8 Palmitic acid, C 16 H^O^ Stearic acid. C^H M O V Oleic acid. C^H^C OXIDATION PRODUCTS OF THE PARAFFINS. 45 Oleic acid differs slightly frcm the other two in that it con- tains two unsaturated carbon atoms C = C. The esters of these acids with the triatomic alcohol glycerin are the ordinary fats of the body. The combination occurs as follows : H 9"~Gd5 H| O 0).(CH 2 ) ie .CH 3 H 2 C-0-CX>(CH 2 ) 16 .CH, H V~ $EK ^H|-0-CO.(CH 2 ) 16 .CH 3 = HC-O.CO.(CH 2 ) 16 .CH S +3H 2 H 2 C loH. Hi O Ca (CH 3 ) 16 -CH 3 H 2 C O CO- (CH 3 ) 16 -CH 8 Glycerin -f 3 Btearic acid, = Tristearin -f 3 water. C S H 6 (OH) 3 + 3HSt. = C a H s St 3 + 3H 2 O. Tripalmitin and triolein are formed in the same way. The fats of the animal body are composed of these three substances mixed in varying proportions. The greater the proportion of olein the lower the melting point of the fat. Unsaturated compounds have a lower melting point than their corresponding saturated compounds. Olein therefore is a fluid at ordinary temperature whilst stearin and palmitin are solids. Soaps. Free fatty acid treated with an alkali or alkaline base will combine with it to form a soap. Example. COOK 16 + KOH = (CH 2 ) 18 -f H 2 f^TT ^-f-3 Potassium stearate (a soap). Soaps are compounds formed by replacing the hydrogen of a fatty acid by a metal. Soaps of the alkali metals are soluble in water. Others, calcium soap, lead soap, etc., are insoluble. On boiling fat with an alkali the latter will drive off the glycerin and form a soap (saponification) C 3 H 5 St 3 + 3KOH = 3KSt -f C 3 H 5 (OH) 3 . Stearin -f Pot. hydroxide = Pot. stearate + Glycerin. 46 OUTLINES OF PHYSIOLOGICAL CHEMISTRY. It is worth while to distinguish between the fats and the waxes. The fats are the esters of glycerin and a higher monobasic acid. The waxes are the esters of a higher monobasic alcohol and a higher monobasic acid. Or diagram matically, Triatomic alcohol (glycerin). 3 (Higher fatty acid). Higher monoatomic alcohol up to C 30 . Higher fatty acid up to C 18 . Fats. Waxes. The fats and oils are chemically alike. A fat when melted is called an oil, and a solidified oil a fat. The true oils have of course nothing in A common with the mineral oils which are hydrocarbons and should, properly speak- ing, be called paraffins. A .Ether- B TVater FlG. 4. TESTS FOR FATS. A. Staining reactions. 1. Sudan III. is a red stain which colors all fats but no other substances. It is in- soluble in water, so has to be used in alco- holic solution. 2. Osmic acid stains olein black, but does not affect other fats. Since animal fats always contain more or less olein, osmic acid is useful for their demonstration, although for general purposes is not so reliable as Sudan III. It is generally used in 1 per cent, aqueous solution. OXIDATION PRODUCTS OF THE PARAFFINS. 47 B. Ether. The fats and free fatty acids (not the soaps) are soluble in ether, so that this is much used as a means of extract- ing fats. The tissue or whatever else it may be, is shaken well with ether in a test-tube there are many forms of apparatus for doing this on a large scale and more thoroughly and the ether then separated from the other material in a separating funnel (Fig. Jf) ; the ether containing the fat in solution rises to the top. The ether is collected in a porcelain dish, allowed to evap- orate and the residue tested for fats, by staining and other reactions which need not be mentioned here. CARBOHYDRATES. Carbohydrates. Mono- saccharides (simple sugars). Disaccharides (compound sugars). Poly- saccharides (starches). The carbohydrates are normal chains of C atoms containing H and O in the proportion of water. Empirical formula of a carbohydrate C n H 2j .O x . 1. Monosaccharides. The sugars are aldehydes or ketones with the remaining C atoms in the chain oxidized to alcohol (OH). For convenience the sugars are all called by names ending in ose with a prefix indicating the number of C atoms. The simplest possible sugar is a biose. O=CH H,COH HCOH H 3 COH H 2 COH Biose. Triose (aldose). Triose (ketose). and so on, tetrose, pentose, hexose, etc. 48 OUTLINES OF PHYSIOLOGICAL CHEMISTRY. The only monosaccharides which occur in nature are certain pentoses and hexoses, of which the following are the best known. HC=O HC=0 )HOH CHOH IEOH CHOH }HOH CHOH ,OH CHOH Xylose (pentose-aldose). CH Z OH: Glucose (hexose-aldose). CH 2 OH C=O CHOH CHOH CHOH CH 2 OH Fructose (hexose-ketose). The monosaccharides occurring in nature have all been syn- thesized in the laboratory and in addition to these a number of other pentoses and hexoses, besides trioses, tetroses, heptoses, octoses and nonoses. About 12 natural sugars are known and over 50 have been synthesized. A glance at the graphic formula of a sugar will show that there are a large number of asymmetrical C atoms, and the number of possible combinations amounts to 16 with the hexal- doses and 8 with the hexketoses. There are therefore 24 possible optically active hexoses besides the racemic forms. This can be easily verified by con- structing graphic formulas, and it is worth while to do it. The aldoses can be oxidized to acids. From glucose for instance three acids can be obtained. Glyconic acid. )H Saccharic acid. )H Glycuronic acid. OXIDATION PRODUCTS OF THE PARAFFINS. 49 Gly conic and glycuronic acid being, like the aldoses, asym- metric molecules with four asymmetric C atoms, each have 16 optical modifications. But saccharic acid is a symmetric mole- cule, so some of its forms are meso. The meso forms are : and the three forms which are the reverse of these, so there are three meso forms and ten optically active forms, 13 space isomers. If glucose is oxidized in the laboratory the aldehyde group is first acted upon and gly conic acid is obtained. Further oxi- dation affects the alcohol group at the other end and saccharic acid is produced. The chain cannot be oxidized beyond this stage without breaking up. In the body the aldehyde group appears to be protected in some way, so that the alcohol group at the other end is first oxidized and glycuronic acid is formed. On further oxidation in the body glycuronic acid breaks up. Glycuronic acid ap- pears in the urine under conditions which will be explained later on. The aldoses by virtue of their aldehyde group will reduce copper oxide (Fehling) as already described for aldehydes. It was said that this is an aldehyde and not a ketone reaction but a ketone containing a number of alcohol groups will react, so that the ketoses will reduce copper oxide or bismuth oxide as well as the aldoses. 4 50 OUTLINES OF PHYSIOLOGICAL CHEMISTRY. Other sugar reactions will be discussed in Chapter V. 2. Disaccharides. A disaccharide is a combination of two sugars with loss of H 2 O. The empirical formula of a monosac- charide being C 6 H 12 O 6 , that of a disaccharide is C 12 H 22 O n . The more important disaccharides are : Saccharose which is glucose -f fructose, Maltose which is glucose -f glucose, Lactose which is glucose -f- galactose. Maltose and lactose will reduce Fehling's solution but saccha- rose (cane sugar) will not. The reason for this is that the latter is so constructed that the aldehyde group of the glucose and the ketone group of the fructose both enter into the combination, whilst with the two former one aldehyde group is left free in each case. To illustrate this the construction of saccharose and maltose is given, (A) HC=0 CHOH CHOH EOH OH , (A) Maltose, one free aldehyde group. CH 2 OH CHOH HC 0- CHOH [OH OH OH Saccharose, no free aldehyde or ketone group. The aldehyde and ketone groups are marked A and K. 3. Polysaccharides. A number of monosaccharide or disac- charide molecules may be combined together to form polysac- charides and each time a fresh monosaccharide or disaccharide is added there is loss of H 2 O so that the empirical formula of a polysaccharide is (C 6 H 10 O 5 ) n . The polysaccharides are very OXIDATION PRODUCTS OF THE PARAFFINS. 51 complex bodies and the number of sugar molecules which they contain is not known. The principal polysaccharides are : 1. Cellulose. Fibrous and woody parts of plants. 2. Starch. Keserve material in seeds, roots and other parts of plants. 3. Glycogen or animal starch. 4. Dextrins. Bodies intermediate between any of these three and sugars. Reactions of polysaccharides with iodine (LugoFs solution). Starch gives a blue color. Glycogen gives a reddish brown color. Dextrins (a) a red color (erythrodextrin). (6) no color (leucodextrin). The di- and poly-saccharides on boiling with dilute mineral acids, H 2 SO 4 is generally used, are decomposed into monosaccha- rides by a process of hydrolysis. The acid acts as a catalyzer. Hydrolysis is the breaking down of complex into simpler molecules with addition of water. I i = ' 1 O-H + H- -f-HJOH It must have been already noticed that H 2 O plays a con- spicuous part in reactions. Two molecules may condense into one with loss of H 2 O " Dehydration." One molecule may split into two with addi- tion of H 2 O " Hydration " or " Hydrolysis." On building up the starch molecule from a monosaccharide there is dehydration, and on breaking it down into its com- ponent monosaccharides there is hydrolysis. Cane sugar on boiling with acids is hydrolyzed to glucose and fructose. 52 OUTLINES OF PHYSIOLOGICAL CHEMISTRY. The more complex insoluble starch molecule is hydrolyzed by a succession of steps through the dextrins and the disaccharide maltose, to glucose. The process is precisely similar to that which is carried out by the enzymes or ferments, amylase, and maltase, and is discussed more fully in Chapter VII. HYDROLYSIS AND SAPONIFICATION. We may digress a little here to point out the differences between hydrolysis and saponification. The terms are only used for reactions with organic compounds. Hydrolysis is simplification with absorption of water. Saponification is simplification with absorption of a hydroxide. Hydrolysis may take place at the point of junction between any two molecules forming a complex, whether alike or not, whether acid, basic or neutral, since the agent of hydrolysis, H 2 O, is neutral. Saponification can only take place when one of the molecules forming the complex is an organic acid, since the agent of saponification KOH or other hydroxide, is a base and can only react with an acid. 1. Examples of hydrolysis. Glycerin / radical ~O-St. + 3H 3 O \)-St. glycerin -f 3 (stearic acid). Examples of saponification. O-St. Fat o Glucose O glucose , TT s\ * radical radical "* **** Maltose = glucose + glucose. CH 3 .qp 3. \> + H 2 CH 3 -CH 2 (Ethylacetester) = CH S COOH + CH 3 CH a OH, (acetic acid + alcohol). PITT C*T1 v^il 3 v>XZ 2 4. ;0 + H 2 CH 3 CH 2 Ethylether = CH 3 CH a OH + CH 3 CH a OH. r O-St. = glycerin + 3 (potassium stearate, a soap). glucose O glucose , M ri^oi ~o,i;oi "T radical radical s= no reaction. CH,.qo ^>0 + KOH CH 3 CH 2 = CH 3 COOK + CH 3 CH 2 OH, (potassium acetate + alcohol). CH 3 CH 2 ">0 + KOH CH 3 CH 2 = no reaction. OXIDATION PRODUCTS OF THE PARAFFINS. 53 Other hydroxides, NaOH, C&(OH) V Ba(OH) 2 , etc., saponify equally well. We may have calcium soaps, lead soaps and so on. Any esters can be saponified (example 3) although the resulting compound may not be what we commonly regard as a soap. The terms however are somewhat loosely used. The organic chemist seldom makes any distinction between hydrolysis and saponification, the latter term being often used to describe a process which is in reality hydrolysis. For instance the " saponification " of fats by superheated steam, or of an ester by the action of an acid. In each case an acid, not a salt, is one of the products, so that strictly speaking this is hydrolysis. CHAPTER IV. A. HALOGEN DERIVATIVES OF CARBON COMPOUNDS. IF CH 4 is treated with chlorine gas it displaces one or more of the H atoms, according to the amount of chlorine used. H H H Cl Cl I I I I I H-C-H H-C-C1 H-C-C1 H-C-C1 C1-C-C1 I I I I I H H Cl Cl Cl Methane. Monochlor- Dichlor- Trichlor- Tetrachlor- methane. methane. methane methane, (chloroform). Similar compounds are formed with bromine and iodine. Chloroform is trichlormethane CHC1 3 , lodoform is triodomethane CHI 3 , Bromoform is tribromethane CHBr 3 . lodoform can be made from alcohol on addition to the latter of a solution of iodine in NaOH and heating. C 2 H 6 OH + 6NaOH + 4I 2 = CHI S + HCOONa + 5NaI + 5H 2 O This reaction is used as a test for alcohol in solution, the merest trace of iodoform being perceptible by its odor. If alcohol is present in appreciable quantities iodoform separates out as a yellow precipitate. B. SULPHUR COMPOUNDS. S and O are' interchangeable in a variety of compounds. The interchange % may take place in the alcohol (OH) groups of oxidized carbon compounds with formation of mercaptans. H 2 COH H 2 CSH + H 2 CH 3 CH 3 Ethanol. Thioethanol (ethylmercaptan). 54 NITROGEN COMPOUNDS. 55 Methyl, propyl, etc., mercaptans can be formed in the same way. The mercaptans are recognizable by their garlic odor, and the above reaction may be used as a test for alcohols. Direct H 2 S however cannot be used. The reaction is obtained indirectly as follows : 5C 2 H 5 OH + P 2 S 5 = 5C 2 H 5 SH + P,O 6 . C. NITROGEN COMPOUNDS. The protoplasm of the living body yields fats, carbohydrates and proteids. The fat and carbohydrate molecules, already discussed, contain no N, but all proteids, their modifications and derivatives contain it, so that a consideration of the com- binations of nitrogen and carbon is in fact an introduction to the study of proteids, and therefore of special importance to the physiological chemist. Nitrogen (N) has three or five valences. In the latter case one of the attached groups is usually OH, or represents OH. N = H 3 H-0-N = H 4 C1-N=H 4 . Ammonia. Ammonium hydroxide. Ammonium chloride. INTRODUCTORY. Carbon dioxide gas, CO 2 , in solution takes up H 2 O to form carbonic acid, CO(OH) 2 . Ammonia, NH 3 , in solution takes up H 2 O to form ammonium hydroxide, NH 4 OH. Carbonic acid and ammonium hydroxide will combine together to form ammonium carbonate and water. H O C O NH 4 H 4 N O C O NH 4 Monoammonic carbonate. Diammonic carbonate. 56 OUTLINES OF PHYSIOLOGICAL CHEMISTKY. The OH of monoammonic carbonate may be replaced by other groups, a chain of C atoms for instance, and the formula may be written as K C O NIL B, being any organic radical. Suppose R is CH 3 , we then have CH 3 COONH 4 or ammonium acetate. This may also be called methylammonium carbonate. If R, COONH 4 is deprived of H 2 O we have JL -NH 2 an amid. If R C NH, is deprived of H 2 O we have R C= N a nitril or cyan compound. The difference between nitrils, amids, and ammonium car- bonate is merely a matter of less or more water in combination, so that they are evidently closely related. NITRILS. Hydrocyanic acid H C = N, Isohydrocyanic acid H N = C. On oxidation : Cyanic acid H - O - C == N, Isocyanic acid H N = C = O, Examples of salts. Potassium cyanide K C = N, Potassium cyanate K O C = N. NITROGEN COMPOUNDS. 57 The highly poisonous nitrils or cyan compounds readily take up sulphur, forming with it harmless so-called rhodanid com- pounds, or more commonly sulphocyanides. HCN + S = H - S - CN. Thiocyanic acid. Thiocyanic acid itself is somewhat poisonous, but its salts are not. Isothiocyanic acid H N = C = S bears the same relation to isocyanic acid as thiocyanic acid does to cyanic acid. In each case S takes the place of O (cf. mercaptans). The isocyan compounds, isomers of the cyan acids, are also called acids but they are not true acids. They do not form stable salts with bases, but enter into combination with certain other substances in a characteristic manner. They will be referred to again. B, C = N readily attaches itself to an aldehyde group and by this means sugars have been synthesized. Starting with glycerin the following graphic formulas explain the process : HO)H oxidized to CHOH -f HCN = CHOH CZ 2 OH CH 2 OH Triose. o=cox CHOH CHOH + 2H 2 = CHOH H 2 O = CHOH CH 2 OH CH 2 OH Ammonium butyrate or propantriol Butantriol ammonium carbonate. amid. O=COH CHOH + HNO 2 = CHOH OH CH 2 Gfl Butantriol amid. Butantriol monacid. 58 OUTLINES OF PHYSIOLOGICAL CHEMISTRY. reduced to H 2 OH Butantriol monal Tetrose. A triose has been converted into a tetrose, and by the same process a tetrose can be converted into a pentose, a pentose into a hexose. Sugars have been synthesized in this way up to a nonose. It has to be taken for granted that chemists can reduce, oxidize, hydrate, dehydrate, etc., at will within certain limits. To enter into details of the methods employed would be out of place here. AMIDS, AMINS, AMINO ACIDS. 1. Amids. An amid is the result of a reaction between the OH group of an acid and NH 3 . Example : 0=C-NH, L + H ,O v>n.j Acetic acid, CH 3 - COOH Acetamid, CH 3 - CONH a 2. Amins. An amin is the result of a reaction between the OH group of an alcohol and NH 3 . Example : H 2 C-!0-H CH, Ethylalcohol, CH 3 - CH 2 OH Ethylamin, CH S - CH a NH a NITROGEN COMPOUNDS. 59 3. Amino (amido) adds. An amino acid is the result of a reaction between the alcohol (OH) group of an oxyacid and NH 3 . Example : ZHl COOH Oxyacetic acid, CH,OH- COOH. Aminoacetic acid, CHNH t - COOH. These reactions are intended merely to show the structure of the compounds and do not represent the methods by which they are formed in the laboratory. The prefix am always denotes an KH 2 group. For an = NH group the prefix im is used. Thus we may have imids and imins. How these arise will become clear as we proceed. 1. AMIDS. An amid may be formed from any of the organic acids. Example : COOH CONH, L, +NH '= Ik +H Ethan acid (acetic acid). Ethan amid (acetic amid). and similarly for the higher homologues, propanamid, butan- ainid, etc. But the most important for us is the diamid urea. Both it and the monamid carbamic acid may be formed from the ammonium carbonates with loss of H 2 O. H-O-C-O NH 4 H 2 O= HOC NH, Monoammonic carbonate, H(NH 4 )C0 3 . Carbamic acid, HCO 2 NH t . ? ft H 4 N-O C-O NH 4 2H 2 O= H 2 N C NH, Diammonic carbonate, (NH 4 ),CO 3 . Carbamid (urea), CO(NH a ),. 60 OUTLINES OF PHYSIOLOGICAL CHEMISTRY. Urea was the first of the organic compounds synthesized. Wohler accomplished it in 1828 by the following reaction. H N=C=O -f NH^OH = NH 4 N=C=O + H a O Isocyanic acid + Ammonium hydroxide = Ammonium isocyanate. But as already mentioned isocyanic acid does not form stable salts, so ammonium isocyanate falls to pieces and reconstructs itself as its more stable isomer urea. It will be seen that the empirical formula of each is CH 4 N 2 O. BIUEET. 1. On melting crystals of urea by heating, a part of the molecules condense with loss of NH 3 forming the soluble biuret. ff -ff fl 7 CiraErnB = H a NC-N- = Urea + Urea. Biuret, C a H 8 N a O a . On treating a solution of this with NaOH and a very dilute solution of CuSO 4 it gives the well-known biuret reaction, a pink to violet color. Proteid material gives the biuret reaction, although proteid contains no biuret. It contains however a radical which is so closely allied to biuret that it affords the same reaction, but this will be discussed more in detail later on. 2. Another part of the urea molecules condense still further to an insoluble compound, cyanuric acid. * I \ -^.-rrr NH [ H N-C/" 5 X) -< Mffi NITROGEN COMPOUNDS. 61 The latter compound is unstable and rearranges itself as 7T H HoCu -}-H 2 S=CuS-f 2(NH 2 -CH 2 . COOH) NH 2 -CH 2 CO The insoluble CuS falls as a precipitate, leaving the amino acid in solution. SEPARATION OF AMINO ACIDS FROM EACH OTHER. The amino acids very readily form ethylesters. CH 3 CH 2 OH CH + = >0 +H 2 NH 2 .CH 2 .COOH NH 2 -CH 2 Ethy] aminoace tester . NITROGEN COMPOUNDS. 67 The ethylesters of the amino acids all have well-defined boil- ing points so can be separated by fractional distillation. But being very unstable the distillation must be carried on "in vacuo " at low temperatures or the esters will break up. Certain characteristics of the amids, amins, and amino acids may be considered side by side for purposes of comparison. 1. Reaction. The amids are neutral. A base NH 3 combines with the OH of an acid (cf. mineral salts). The amins are basic. There is no acid O in the molecule to neutralize the base NH a . The amino acids are both acid and basic. Since neither the acid nor the base are in direct combination each of them retains its own characteristics. The amino acids are acid by virtue of their COOH group and at the same time basic by virtue of their NH 2 group. 2. Action of Nitrous Acid. Nitrous acid (HNO 2 ) acts in the same way on all NH 2 groups whether amid, amin, or amino. It has the power of substituting OH for the NH 2 group, at the same time breaking up the latter and liberating free N. The reaction occurs as follows : ff ! I ff K-C-iNjH 3 =K-C-< -0-H+N a +H 2 + HOiNlO Amid+niteous acid, Acid, K-CONH a +HN0 2 . R-COOH+N a +H 2 0. K CH, JNJH 2 = E CH 2 OH+N 2 +H,O + HO JN JO Amin. Alcohol. 68 OUTLINES OF PHYSIOLOGICAL CHEMISTRY. COOH COOH 3 HC- - j N j H, = HOOH +N 2 +H 2 O E+HOJNiO K Amino acid, R CHNH 2 COOH. Oxyacid, R CHOH- COOH. HNO 2 is unstable, passing off quickly into NO and NO 2 gases (brown fumes) with loss of H 2 O ; so has to be used in a nascent condition. To obtain this potassium nitrite, KNO 2 , is added to the solution to be tested and then H 2 SO 4 : 2KN0 2 + H 2 S0 4 = K 2 S0 4 + 2HNO 2 . The liberated HNO 2 immediately attacks the NH 2 groups. It is its desire to get rid of its OH groups which is the cause of this. If left to itself: o + 0==0 (N a O 2 usually called NO.) (N a 4 usually called N0 2 .) and goes off in brown fumes, but it is able to get rid of its OH more quickly by attacking NH 2 groups. If, however, HNO 2 comes in contact with an NH (secondary or imid group) the action is different. >NiH E C v R-N-N=0 +H 2 -fON|OH Secondary NH group. Nitroso combination. The HNO 2 parts with its OH which combines with the single H to form H 2 O, and the two ISPs combine with each other. In this case there is nothing left for the O, so it remains attached to the N and a so-called nitroso compound results. The nitroso = N N = O reaction is often of value for the detection of NH groups. HNO 2 has no effect on a triamin. NITROGEN COMPOUNDS. 69 E C\ E-CAN K-C/ having no H cannot assist HNO 2 to get rid of its OH, so the latter goes off in brown fumes in the ordinary way, leaving the triamin untouched. Sodium hypobromite, NaOBr, in alkaline solution will also split off N from NH 2 groups, though not so completely as HNO 2 . About 90 per cent, of the N may be obtained in this way. The reaction with urea occurs as follows : 1+iOiNaBr -j-NaBriO /iN|H 2 i |O=C\ ! ^U j =C0 3 +N 2 +2H 8 0+3NaBr N: 1> l-tlj; j 1+iOjNaBr This test is simple and much used in clinical pathology for estimation of the amount of urea in urine. But it is not accu- rate enough for quantitative analysis and the scientific chemist leaves it severely alone. The test is made in a fermentation tube and the amount of gas evolved measured as N, the CO 2 being absorbed by the alkali to form carbonates. OTHER AMINO ACIDS. Besides the monoamino-monobasic acids the proteid molecule contains a small amount of: 1. Monoamino-dibasic Adds. Of these the most important are : COOH !OOH CHNH, !HNH a CH, 2 CH, COOH COOH Amino butandiacid, asparaginic acid. Amino pentandiacid glutaminic acid. 70 OUTLINES OF PHYSIOLOGICAL CHEMISTRY. These contain two COOH groups to one NH 2 , so are dis- tinctly acid. Asparagin is sodium asparaginate COONa . CHNH 2 . CH 2 . COONa. 2. Diamino Monobasic Acids. !OOH HNH, H 2 H 2 H 2 ._,-.-, H 2 NH, Diamino pentanacid Diamino hexanacid o, 8, diamino valerianic acid , e diamino caproic acid (ornithin). (lysin). These contain two NH 2 to one COOH, so are distinctly basic. It will be observed that, like leucin, all the above are alpha amino acids. In addition ornithin contains NH 2 in the delta, and lysin in the epsilon position, so they are called a, 8 amino valerianic acid and a, e amino caproic acid respectively. GLYCOSAMIN. Sugar occurs in combination in the proteid molecule as glycosamin. CHO . CHNH 2 . CHOH . CHOH . CHOH . CH 2 OH. Glycosamins can occur as insoluble polymers (cf. glucose and glycogen) and in this form, called chitosamin, mixed with lime salts, constitute the hard chitinous covering of lobsters and other crustaceae. Chitosamin does not occur in vertebrate animals. Their bones are modified proteids (gelatin, collagen) mixed with lime salts. Cholin. Before leaving the subject of the N compounds the constitution of cholin may be given. NITROGEN COMPOUNDS. 71 Cholin is trimethyl oxethyl ammonium hydroxide. Knowing this it is easy to construct its graphic formula : Ammonium hydroxide is j X-0-H Three of the hydrogen atoms react with CH 3 OH, making : CH CH, ^>N OH + 3H,O CH, H The remaining H reacts with ethandiol (oxethyl alcohol) (glycol). CH a OH CHjOH+H-NsCCHj), = CH a OH CH, N=(CH 8 ) 8 +H,O Cholin. P COMPOUNDS. Phosphorus occurs in combination in the proteid molecule in the shape of lecithin. Lecithin is diolein or dipalmitin or distearin cholin phos- phoric acid and with the knowledge already gained its formula can be constructed. Taking the distearin form as an example : H-CK H 0-P= Phosphoric acid. HC O St H 2 C O St J Y Distearin + Phosphoric acid. Distearin phosphoric acicL 72 OUTLINES OF PHYSIOLOGICAL CHEMISTRY. H 2 HCOSt H 2 COSt if' CH 2 = HCOSt <> CH 2 +H 2 O CH 2 OH H 2 COSt H CH 2 OH Distearin phosphoric acid + Cholin Lecithin. This leaves still a free OH group on the phosphoric acid by which it can attach itself to some other component of the pro- teid molecule with loss of H 2 O. The reason for calling lecithin a phosphorized fat is made clear by a knowledge of its constitution. CHAPTER V. CYCLIC COMPOUNDS. I. THE chains of C atoms have a tendency to curl over and join at the two ends, forming in this way a closed chain. In order that the chains may close up, there must be groups at either end which would naturally have a tendency to combine with each other. An acid COOH group would have a tendency to combine with an alcohol to form an ether, or with an NH 2 group to form an imid, NH. A chain therefore with COOH at one end and NH 2 at the other would be likely to close up if the chains were bent over sufficiently so that the two would come together. The shorter chains, amino acetic acid, amino propionic acid, are not able to bend over sufficiently to meet. But 7 amino butyric acid is long enough, and the same may be said of all amino acids where the NH 2 group is above /3. If an alcohol group joins with a COOH group the compound is called a Lactone, and COOH combining with NH^ a Lactam. H. +H,0 H 2 C CH 2 j..OH_J H,C y oxybutyric acid. y oxybutyric lactone (an autoester). H 2 C CO H 2 c-cooH y^ H a C-CH 2 NH, H 2 C-CH, y amino butyric acid. -y amino butyric lactam (an auto aminester). 73 74 OUTLINES OF PHYSIOLOGICAL CHEMISTRY. II. Again two or more separate molecules of the same sub- stance may condense with loss of H 2 O or NH 3 and form a closed chain. Example, cyanuric acid (page 60). III. Two or more similar unsaturated compounds may inter- change a valence and thus form a closed chain. Ethine (acetylene gas) is such a compound HC == CH. If three molecules of ethine are arranged as follows ; CH % (2) (Dl CH CH CH * (3) CH It can easily be seen that if (1) gives up a valence to (2), (2) to (3) and (3) to (1) the resulting molecule will appear as, This, by far the most important of the cyclic compounds, is a benzene ring, and on the benzene ring, as a basis, an infinite variety of molecules can be built up. Benzene may combine with an acid. In the case of an alcohol combining with an acid to form an ester there is always an O between the two molecules. -C-NH CH 2 - COOH +JHJHN-CH 2 -COOH = \/ +HC1 Benzoyl chloride + glycocoll. = Benzoyl glycocoll aminester. The glycocoll has been benzoylized. In benzoyl glycocoll aminester, hippuric acid will be recog- nized again. It can therefore be produced in the laboratory as well as in the body. Phenylisocyanate is used for the same purpose as benzoyl chloride. It affords more definite compounds than benzoyl chloride, so is more valuable for accurate work, but its expense prevents its use in the laboratory for ordinary purposes. The reaction with NH 2 groups may be given, taking glycocoll again as an example. -N=C=O /\ _ NH C NH CH 2 COOH +NH 2 .CH 2 -COOH = yj Phenyl isocyanate + glycocoll. Phenyl glycocoll isocyanate. It will be observed that in this case there is no loss of water. The C of the N = C = O group detaches one valence from the N to join to the NH 2 group, and the H thus liberated attaches itself to the first N and saturates it again. _ _ o ] %^ I = E-NH-C-NH-R H -NH-E 88 OUTLINES OF PHYSIOLOGICAL CHEMISTRY. A curious point about the aminisocyanates may be brought out by substituting H for the radical on either side of the isocyan group. R iNH NH|-E makes H 2 N C-NH a JH HJ or urea, so that we may regard the isocyanates as merely sub- stituted urea. PHENYL HYDRAZIN, H 6 C 5 . NH. NH 2 . The NH 2 very readily gives up its two free H atoms to an aldehyde or ketone group, the H atoms attaching themselves to the aldehyde or ketone O to form H 2 O and the*N to the C by two valences j/^j-NH-NiH.+okCH i/^-NH N=CH ' k = I I +H Phenyl hydrazin' -f ethanal, = | Phenyl ethyl hydrazone, C 6 H B - NH- NH 2 . C 6 H 6 - NH- N- C a H 4 . This reaction is much used as a test for aldehydes and ketones, the resulting hydrazones, as they are called, crystallizing easily. ^^^ The osazone test for sugars is based on this reaction but here the process is more complicated. If to a solution of a hexaldose (glucose) phenylhydrazin is added and the mixture warmed, three successive reactions occur. "KTTT TVT'TT t /\> /">TT / > /"^TIT/"\TT\ /"ITT /~\TT , IsJti IS:ri 2 -(-(J.=:Cil 2 (CHOHj^ Cli 2 OH - ! = /^j NH N=CH ( CHOH)< CH 2 OH +H 2 O \/ Phen yl hydrazin + Glucose = Phenyl glucose hydrazone. (2) A second molecule of hydrazin now attacks the group lying next to the aldehyde group, depriving it of its two hydro- CYCLIC COMPOUNDS. 89 gen atoms ; at the same time forming anilin with release of NH 3 . The CHOH group of the sugar thus attacked has only an O left and becomes a ketone. HC=N-NH C 6 H 5 /\ HC=N - NH OH) 8 (CHOH), 2 OH CH 3 OH Phenyl hydrazin + Phenylglucosehydrazone Intermediate body. (3) The ketone thus formed is immediately attacked by a third phenylhydrazin molecule as follows : HC=N - NH C 6 H 6 HC=N NH (CHOH) 3 : 2 OH Intermediate body + Phenyl glucose hydrazone = Glucosazone (an osazone). "With ketoses the ketone C = O group is always next to one of the end CH 2 OH groups, so that the ketone group of a ketose may be regarded as holding an alpha position. Phenylhydrazin brought in contact with a hexketose (fruc- tose) first forms a phenylketosehydrazone with the ketone group. A second molecule of the phenylhydrazin then deprives the adjacent CH 2 OH group of its hydrogen atoms, forming anilin and NH 3 . This newly formed aldehyde group then reacts with a third molecule to form the osazone. Thus, although in this case we start with a ketose (fructose), the final product is precisely the same as with an aldose (e. g., glucose). This can be easily verified on working out the reaction, with the help of graphic formulas. In each case a definite crystal- lizable substance with a definite melting-point is obtained, but 90 OUTLINES OF PHYSIOLOGICAL CHEMISTRY. the two substances may be precisely alike, providing their re- maining H and OH groups are arranged in the same way. Where these differ the osazones will differ slightly in solubility, so that to a limited extent monosaccharides may be differenti- ated by means of their osazones. DOUBLE KINGS. To the benzene ring a second ring may be attached to form naphthalene, and this may be oxidized to naphthol. \/OH OH Naphthalene, a naphthol, /3 naphthol, C JO H 8 C 10 H 7 OH C 10 H 7 OH On the double ring it makes a difference whether the OH group lies next to the link, as the alphas, or one removed from it as the betas. It makes no difference on which of the four alphas the OH is placed. Its relations to the rest of the mole- cule are the same in each instance, and the same is true of the four beta positions. There are therefore two naphthols, a and ft. MOLISCH'S REACTION FOR SUGARS. If an alcoholic solution of a naphthol is added to a solution containing sugar, or any carbohydrate, and a little H 2 SO 4 then carefully poured down the side of the tube, the latter sinks to the bottom and at the point of junction a violet color at once appears, due to formation of furfurol. This is a very delicate reaction, occurring even if the sugar is in combination. Proteid material containing glycosamin in combination gives it. CHOLESTERIN is a fat-like substance, which however is not a true fat, but a higher monoatomic alcohol, the empirical formula of which CYCLIC COMPOUNDS. 91 is C^H^OH. Since there are not enough hydrogen atoms to form a chain it is supposed there must be a benzene ring in it somewhere, but its constitution is not understood. GLUCOSIDES are glucose combined with some cyclic alcohol to form glucose ethers. Glucosides do not reduce Fehling's solution or form osazones, so it is probable that the aldehyde group enters into the combination. (Cf. Disaccharides, p. 50.) Example : OH OH \ CH 2 OH f\ CH 2 glucose LJ + glucose = I I +H 2 Salicyl alcohol = Salicin. Salicin is found in the bark of the willow. There are a large number of glucosides, but since they are exclusively products of plant life, never being found in animal tissues, they need not detain us. HETEROCYCLIC COMPOUNDS. If the ring or one of the rings contains fewer than six carbon atoms it is irregular and is called heterocyclic. One of the six C atoms may be replaced by N C/\C C/\C C\^C C\Al c IT Benzene. Pyridin. Or the ring may consist of four C atoms with NH, S, or O. Thus: C C, ;C C IB. s 06 Pyrrol. Thiophen. Furan. Furfurol. 92 OUTLINES OF PHYSIOLOGICAL CHEMISTRY. As is the case with the isocyclic, the proteid molecule is poor in heterocyclic compounds. There is in fact only one of importance, a ring of which indol is the best known repre- sentative. Indol has a double ring, of which one is irregular. COH Indoxylsulphuric acid (urine indican). Indol is oxidized to indoxyl in the body and in consequence of the OH group is excreted, like phenol, in combination with sulphuric acid (urine indican). Urine indican must not be confounded with the indican of the indigo plant which is a glucoside (glucose + indoxyl, p. 91). Indol is formed in the large intestine, by the action of bacteria, principally coli communis, as a product of the decom- position of the proteid molecule. Normally the indol, or by far the greater part of it, passes off with the feces, but if there is intestinal obstruction, or an unusual amount of intestinal putrefaction, the indol may be largely absorbed in the body and undergoes the changes m^^ tioned above. Indoxyl is easily oxidized to indigo ^ 11 ! C C l I! :i + 2H 2 /\ '\/CH NH Indoxyl. Indigo blue. CYCLIC COMPOUNDS. 93 As soon as oxidation occurs one link between the CO and CH is released. Two molecules then combine as above, each losing H 2 which combines with O to form H 2 O. Along with the indigo blue a small amount of indigo red is also formed. Indigo red is the same as indigo blue but with one of the indol rings reversed. The two are space isomers. H NH NH CO Indigo blue. Indigo red. Indicanuria or indican in the urine can be experimentally produced in animals by resecting a portion of the intestine and replacing it inversely. In this portion peristalsis is reversed, and there is obstruction. Coli communis and other bacteria are able to multiply at any such point, even if it is in the upper part of the small intestine where they do not normally exist. Indol, therefore, is formed in excess and appears in the urine as iudican. Besides indol, skatol or methyl indol is formed by bacteria in the intestinal tract, and it is to this that the fecal odor is mainly due. / \:OOH TH NH NH Indol. Skatol. Skatol carbonic acid. Skatol carbonic acid is also a product of proteid decomposition by bacteria. xs. CH, X CHNH 2 - COOH H Tryptophan. Tryptophan or skatol amino acetic acid, exists in combina- tion in the proteid molecule. (See postscript.) 94 OUTLINES OF PHYSIOLOGICAL CHEMISTRY. REACTIONS. Indol treated with H 2 SO 4 and KNO 2 gives a red color, due to the production of nitroso indol. H 2 S0 4 + 2KN0 2 = K 2 S0 4 + 2HNO 2 nascent nitrous acid. +H 2 H N N=0 + ON | OH Indol + nitrous acid Nitroso indol. Nitroso skatol and the nitroso .compounds of the other derivative of indol do not afford the red color. Tryptophan if free gives a pinkish color with chlorine or bromine water. Tryptophan in combination in the proteid molecule does not afford this reaction, but it will give a violet color if treated with H 2 SO 4 and acetic aid (Adamkievicz reaction). It has long been known that if the acetic acid has been freshly made it will not give the Adamkievicz reaction. But it has only recently been shown that it is not the acetic acid itself which reacts, but an oxidation product, glyoxylic acid. CH 3 COOH + O 2 = CHO. COOH + H 2 O Acetic acid. Ethanal acid (Glyoxylic acid). Sodium glyoxylate can be easily made by reduction of oxalic acid with sodium amalgam and, since this is stable, it is now used instead of old acetic acid for the test. (Hopkins reaction.) Na.Hg + COOH.COOH = CHO.COONa -f H f O + Hg. Sodium amalgam. Oxalic acid. Glyoxylic acid. The Hg merely serves as a restrainer for the Na, which would react too violently if used alone. On adding H 2 SO 4 the Na is driven off and the glyoxylic acid gives the violet color with the tryptophan. CYCLIC COMPOUNDS. 95 The structure of the colored product is not known. The alkaloids are complicated vegetable products, the con- stitution of only a few being known. There are three rings which chiefly serve as a basis for their make up. N \/ \/ N N Pyridin. Chinolin. Isochinolin. The alkaloids are largely poisonous but do not directly con- cern us except for the fact that at one time they were supposed to be products of plants solely. If, therefore, alkaloids were found in a body " post-mortem," it was taken for granted that death was caused by vegetable poisoning and many a man has been hanged on this evidence. It is now known, however, that this is an erroneous supposi- tion, for many bacteria produce alkaloids and after decomposition has set in alkaloids may be found in animal organs without death having been necessarily caused by them. PUKIN Although the purin bases are cyclic compounds they can hardly be classed with those so far under consideration since they are not based on the condensation of ethine. (1) N=(6)CH HN C=O HN C=O (2) HC (5)C NN(7) HO C NH O=C C NH 6H(8) . PROTEIDES. This group includes : 1. Glycoproteids. Compounds of albumen loosely combined with glycosamin or other carbohydrates. 2. Hemoglobins. Compounds of hematin with globin (an albumen). 3. The Nudeoproteids. Compounds of nucleinic acid with albumen. Summarizing the knowledge thus far gained we find : 1. The albumens. 2. Proteoses. Albumens modified by hydrolysis. 3. Albumenoids. Albumens modified by body cells. 4. Proteides. Albumens -f something else, the albumen imparting the proteid character. All these are included under the general term proteid, but the albumens are obviously the corner stone of the whole edi- fice, so are called " true proteids." In speaking, therefore, in the following pages of the proteid molecule this practically refers to the albumens. ULTIMATE ANALYSIS. The approximate elementary constituents of proteids have already been given. On ignition there is always found a small amount of ash. This varies with the kind and purity of the proteid, but is made up of varying amounts of chlorides, sul- phates, phosphates and carbonates of the metals sodium, potas- sium, calcium, magnesium and iron. Some of this ash is min- eral matter merely mixed with the proteid, whilst a portion is probably combined. There is reason to doubt that a truly ash- free proteid has ever been obtained. Many attempts have been made to determine the molecular weight of albumen and other proteids by means of ultimate THE PROTEEDS. 105 analysis, and by determination of the osmotic pressure of their solutions. No accurate figures have yet been obtained, but it is certain that the molecule is very large, that there is great variation in its size and that it decreases with the progress of hydrolysis, so that albumoses and peptones have a much smaller molecule than the albumens. Some attempts have been made to arrive at a structural formula for the comparatively simple peptones, but even these are so extremely complex that this has not yet been accomplished. Ultimate analysis of proteids therefore has been practically abandoned, but by hydrolyzing or oxidizing the entire molecule of a proteid it can be split up into fragments of a simple nature, whose constitution is known, and it is possible that by a study of these fragments the manner in which they are com- bined to form the proteid molecule may be learned. Oxidation by means of potassium permanganate or other oxidizing agents is unsatisfactory because : 1. The oxidizing process cannot be accurately regulated so that results are not constant. 2. The fragments so obtained probably do not exist pre- formed as such in the original molecule. Hydrolysis, effected by means of boiling with dilute mineral acids, or by treating with digestive enzymes, such as trypsin, at body temperature, is much more satisfactory because : 1. The process can be accurately regulated. 2. The fragments obtained probably exist preformed as such in the original molecule. These fragments combined together go to build up the pro- teid molecule, so they are called its nuclei. The object is to get out the nuclei as nearly intact as possible. It has been said that albumens on hydrolysis yield the proteoses 106 OUTLINES OF PHYSIOLOGICAL CHEMISTEY. as intermediate products. The proteoses are called interme- diate products because, on carrying on the hydrolysis still further, they can be split up into the comparatively simple nuclei. Proteid + n(H 2 O) == Proteoses. Proteoses -f n(H 2 O) = Nuclei. We may now briefly consider : 1. Products of hydrolysis. 2. Products of oxidation. 3. Products of bacterial action. 4. Products of body cells. 1. PRODUCTS OF HYDROLYSIS. Proteids on hydrolysis have been found to yield the follow- ing list of nuclei, though not all of them are found in all proteids. The principal ones only are given. A. Monoamino Acids. 1. Aminoacetic acid (Glycocoll), CH 2 NH 2 . COOH. 2. Aminopropionic acid (Alanin C 3 ), CH 3 . CHNH 2 . COOH. 3. a aminocaproic acid (Leucin C 6 ), CH 3 . (CH 2 ) 3 . CHNH 2 . COOH. B. Amino Diacids. 4. Amino succinic acid (Asparaginic acid C 4 ), COOH.- CHNH 2 .CH 2 .COOH. 5. Aminoglutaric acid (Glutamic acid C 5 ), COOH.CHNH 2 - (CH 2 ) 2 .COOH. C. Diamino Adds. 6. Diamino acetic acid, CH(NH 2 ) 2 .COOH. 7. a, S, diamino valerianic acid (Ornithin C 5 ), (CH 2 ) 2 .CHNH 2 .COOH. THE PROTEIDS. 107 8. a, e, diamino caproic acid (Lysin C 6 ), CHNH 2 .COOH. D. Amino hexose. 9. Glycosamin (C 6 ) CHO.CHNH 2 .(CHOH) 3 .CH 2 OH. E. IsocydiG compounds. 1 0. Phenylaminopropionic acid (Phenylalanin C 9 ), C 6 H 5 .CH 2 .- CHNH 2 .COOH. 11. Paraoxyphenylaminopropionic acid (Tyrosin C 9 ), C 6 H 4 - (OH), .(CH 2 .CHNH 2 .COOH). F. Heterocydic Compounds. 12. Skatol ammo acetic acid. (Tryptophan.) /\ prr ! I f\* I I I X CHNH a - COOH NH G. Unknown Composition. 13. Histidin, C 6 H 9 N 3 O 2 . H. Sulphur Compounds. 14. a amino {3 thio propionic acid (cystein) CH 2 SH.- CHNH 2 .COOH. All the above are amino acids with the exception of glyco- samin (amino aldehyde), but there is another exception of con- siderable importance. 15. Guanidin, CNH(NH 2 ) 2 . Guanidin occurs in combination with ornithin to form arginin with loss of NH 3 , so is not split off on hydrolysis. Arginin is found as the nucleus but is in reality a double (binary) com- pound, Guanidin -f ornithin. Two points may be noted. 1. The nuclei all contain nitrogen. 108 OUTLINES OF PHYSIOLOGICAL CHEMISTRY. 2. The NH 2 is always on the C next to the COOH group, i. e.j the nuclei are alpha amino acids. II. PRODUCTS OF OXIDATION. Oxidation carries the decomposition of the proteid molecule much further than hydrolysis, oxalic acid and gases CO 2 , NH 3 , N and H 2 O being among the final products. Intermediate prod- ucts are more or less complex oxycids about which not much is known. The point to be grasped here is that oxidation attacks the individual nuclei, even though they may be still attached to the main molecule, whilst hydrolysis simply splits up the main molecule, leaving the nuclei intact. III. PRODUCTS OF THE ACTION OF BACTERIA. A third method of attacking the proteid molecule is by means of bacteria. Many bacteria can hydrolyze proteids, e. g., liquefaction of gelatin or blood serum, but their action does not stop here. They have a way of splitting off the CO 2 of the acid group from the amino acids, often leaving the rest of the nucleus intact, for a time at any rate. Ornithin and lysin are often acted on in this way. CH 2 NH 2 (CH 2 ) 2 CHNH 2 - COOH = CH 2 NH 2 (CH 2 ) 2 CH 2 NH 2 + CO, Diamino valerianic acid Tetramethylendiamin (putrescin) (ornithin). -j- carbon dioxide. CH 2 NH 2 (CH 2 ) 3 CHNH 2 . COOH = CH 2 NH 2 (CH 2 ) 8 CH 2 NH 2 + CO 2 Diamino caproic acid Pentamethylendiamin (cadaverin) (lysin). + carbon dioxide. These and many others formed in a similar way from monoamino acids are the bases which we so often read about as being products of putrefaction. Cadaverin and putrescin now suggest more to us than merely something with a bad smell. THE PROTEIDS. 109 The bacteria may then proceed to partially oxidize these bases, forming oxyamins. The bases and their partial oxidation products resulting from the action of bacteria are called ptomains. Instead of CO 2 , bacteria may split off NH 2 , forming acids instead of amins. Finally the ptomains or other products on further bacterial action are oxidized and broken up into gases and nitrates. These processes are carried on by various groups of bacteria, a fresh group taking up the work where the action of a previous group ceases, these changes occurring naturally in both soil and water. IV. PRODUCTS OF ACTION OF BODY CELLS. Hydrolysis and oxidation go on hand in hand in the body, the final products being urea, CO 2 and H 2 O. Intermediate products which do not leave the body as such are called leuco- mains. The leucomains are analogous to the ptomains. Summarized in tabular form : Method. Process. Intermediate Products. Final Products. Acids or enzymes. Oxidizing agents. Bacteria. Body cells. Hydrolysis. Oxidation. Hydrolysis -{- Oxidation. Hydrolysis -(- Oxidation. Xone. 1 Oxyacids. Ptomains. Leucomains. Amino acids, nuclei. Oxalic acid, gases, water. Xit rates, gases, water. Urea, gases, \vater. Or diagrammatically, the numbers indicating various nuclei linked together 1 The earlier intermediate proteoses are not taken into consideration. 110 OUTLINES OF PHYSIOLOGICAL CHEMISTRY. LlriK, :Lfrikr 11 12 11 Proteid molecule. H20 <^0 ^0 HsO .HjO HsO tt.0 ^ i i 1 : t 3 J 1 1 3 1 j 2 1 1 1 J ) : i t Hydrolysis. ^5> j 1 8 I - CHNH,..COOH But in this way the COOH groups are neutralized whilst the basic NH 2 groups remain free, a combination which would lead to a strongly basic character for the compound molecule. In order to preserve the weak amphoteric reaction we must suppose that the NH 2 groups enter into the reaction and are 112 OUTLINES OF PHYSIOLOGICAL CHEMISTRY. neutralized as well as the COOH groups. In this way we can build up the complex as follows : 2. Combination OC N. HN 2 - CH a OC-[o^+HJ_NH CH 2 oc i" o H+H"] NH COOH =H 2 N- CH- C0 NH. CH- CO- NH- OH- CO- NH- CH- COOH At one end there is the basic NH 2 and at the other the acid COOH, so that the acid and basic qualities are precisely the same as in the original nuclei, and remain the same, no matter what the size of the complex may be. This constitutes a glycocoll chain, but remembering that our nuclei are alpha amino acids, R CHNH 2 .COOH, we can substitute any of them instead of glycocoll, and in this way build up a part of the proteid complex as follows : OH:H OHIH OHH H 2 N- CH- CO ^NH- CH- CO ; NH- CH- CO NH- CH- COOH I I (CH 2 ) 2 (CH,), CH, C 6 H 4 (OH) COOH CH 2 NH 2 Leucin. Tyrosin. Glutamic acid. Lysin. The dotted lines show where combination took place and where splitting would occur on hydrolysis. 1. Hydrolysis. The nuclei are split off as such. 2. Oxidation. The side chains may be supposed to become oxidized down until we get THE PROTEIDS. 113 H 3 N- CH- CO-NH- CH- CO- NH- OH- CO NH- CH- COOH COOH COOH COOH COOH The next step would be to split off CO 2 and oxidize the CH groups of the main chain into H,N- CO- CO- NH- CO- CO- NH- CO- CO- NH- CO- COOH But such a combination would be so unstable that it would hydrolyze of itself, without any stimulus in the shape of a catalyzer such as a mineral acid or trypsin, into (CONH 2 .- COOH) n oxalamid. Oxalamid again is very unstable and would of itself hydrolyze to oxalic acid, COOH.COOH and NH 3 . Oxalic acid, CO 2 and NH 3 are among the chief products of oxidation by potassium permanganate. The fact helps to support the theory. 3. Hydrolysis + oxidation as in the body. Supposing the side chains oxidized down to OHPH OHH OH;H H 2 N CH - co pra CH co jNH - CH - co JNH CH - COOH COOH COOH COOH COOH If hydrolysis occurs at this point, we get (COOH.CHNH 2 .- COOH) n or amino malonic acid. But this is very unstable, being easily, by slight warming even, split up into glycocoll, CH 2 NH 2 .COOH, and CO 2 . As already mentioned glycocoll is very likely the immediate precursor of urea. There are other arguments which can be used in support of this theory, but they need not be discussed here. It must not, however, be forgotten that this is still a theory. There is no actual proof that the molecule is constructed in this way. Defects of Hydrolysis. Hydrolysis certainly affords a better insight into the make-up of the proteid molecule than oxidation, but even hydrolysis is not perfect. 8 114 OUTLINES OF PHYSIOLOGICAL CHEMISTRY. If carried out by means of boiling with acids, certain dark colored condensation products are afforded, so called melanoidins, which certainly do not exist as such in the proteid molecule, and in the formation of which some of the nuclei must be concerned, so to this extent are lost for observation. On digestion with trypsin melanoidins are not formed, but some of the nuclei may undergo changes. Taking the nucleus represented by the indol ring for example, N/V NH It is probable, for reasons given later, that this exists in the proteid molecule as tryptophau. As hydrolysis proceeds the fluid gives a pinkish color on treating with chlorine or bromine water, showing the presence of free tryptophan. J3ut before the hydrolytic process is complete the tryptophan reactions are no longer given. The ring '-' is still there but no longer as tryptophan. It may appear as -7CH, it X CH 2 NH 2 NH skatol methylamin, CO 2 being lost, or in other forms. Again on hydrolysis of proteids either by acids or trypsin NH 3 is given off. This is probably amid N driven off from the amino- diacids. Whatever its origin, it indicates something which is lost for observation. X THE PROTEIDS. 115 As an example of the different forms which a nucleus may assume according to the way the proteid is treated tyrosin may be taken. 1. Treated with acids, it appears as tyrosin, 6" CH a -CHNH a -COOH and this is probably the form in which it exists in the proteid molecule. 2. With trypsin some of the tyrosin may lose CO 2 and appear as paraoxyphenylethylamin, 3H a -CH a NH 2 3. "With bacteria NH 3 may be lost, and it appears as para- oxyphenylpropionic acid, )H COOH CH 2 -CH 2 - 4. On oxidation it may appear as phenol or cresol. 5. On fusing dry proteid with KOH there is condensation and indol or skatol is formed. CH, L + = f 1 I + CH 2 . COOH OH '\/ Phenol acetic acid. /\ 1 CH 3 - COOH \/\/ Nil Indol acetic acid (Skatol carbonic acid). With bacteria or oxidation. ,/\ CH S OH \/ Methyl phenol (Paracresol). CX/"' NH Methyl indol (Skatol). o0 Phenol. Oj NH Indol. In the urine H0 8 S O | | I i O SO 3 H II Indoxyl sulphuric acid (Indican). Phenol sulphuric acid. Indol is a toxic substance and when absorbed in the intestine is carried by the portal vein to the liver where it is oxidized to indoxyl which combines with sulphuric acid to form indoxyl sulphuric acid which is not toxic and is excreted as a potassium salt in the urine. ^ /\ II K THE PBOTEIDS. 129 Skatol is rendered harmless and eliminated in the same way. This synthesis is one of many similar ones designed to protect the organism from the poisonous effects of proteid decomposi- tion products. Phenol and cresol are also eliminated as potas- sium sulphates. The amount of combined sulphates in the urine is a measure of the extent of intestinal putrefaction. The sulphates in urine are grouped as mineral (uncombined) and combined, the latter being those in combination with indoxyl or some other aro- matic ring. BaCl 2 is added to the urine and this precipitates the uncom- bined but not the combined sulphates. BaCl 2 + K 2 SO 4 = BaSO 4 + 2KC1. The fluid is then filtered and the filtrate boiled with hydro- chloric acid. This splits off the indoxyl or other aromatic compound and the combined become uncombined sulphates which can then be precipitated by BaCl 2 and weighed as BaSO 4 . This method gives an index to the total amounts of aromatic products of intestinal putrefaction which are absorbed, indols, phenols, or cresols. A part of these products are excreted in combination with glycuronic acid, but the amounts are com- paratively insignificant and are ignored in making the test. Clinical Test for indican alone. The urine is treated with an oxidizing agent concentrated HC1 containing a little FeCl 3 ; this oxidizes the indican with formation of indigo blue, which is obtained by shaking out with chloroform, in which it is soluble. The depth of blue color in the chloroform solution affords a rough estimate of the amount of indican present. Indoxyl will oxidize itself to indigo blue by taking up O from the air, but the FeCl 3 being an oxidizing agent, hastens the process. 9 130 OUTLINES OF PHYSIOLOGICAL CHEMISTRY. H. Sulphur Compounds. The S of the proteid molecule exists chiefly as cystin. Cystin is a binary compound made up of two molecules of cystein ; analogous therefore to arginin. E-S jH+Hj S CH 2 TH 2 CHNH a = H COOH a amino, /3 thiopropionic acid Cystein + Cystem Since the two molecules of cystein combine with loss of H 2 , not of H 2 O, cystiu is not split up by hydrolysis but appears as such among the proteids ; not as the ultimate nucleus cystein. To estimate the total amount of S, the proteid is completely oxidized by nitric acid, BaCl 2 added, and the sulphur weighed as BaSO 4 . On boiling some proteids with lead acetate in alkaline solu- tion, black lead sulphide, PbS, is quickly precipitated. The alkali splits off the S, which then combines with the Pb. But there is always some sulphur left which cannot be precipitated in this way. The former is loosely combined and the latter is in stable combination. It was formerly supposed that the loosely combined S exists in unoxidized form K S H, and the stable in oxidized form as sulphites or sulphates, so the S of proteids was classified as oxidized and unoxidized. But there is no evidence to show that oxidized S occurs in the proteid molecule. Sulphites and sulphates are never found among the products of hydrolysis. Moreover experiments with pure cystin have proved that its S is distinctly stable and can only be partially precipitated even by prolonged boiling with alkaline lead acetate. We may conclude, therefore, that the stable S of the proteid THE PROTEIDS. 131 represents the cystin, whilst the loosely combined must exist in some other form as B, S H. Just how it exists is not accurately known, but mercaptans, - methyl, CH 3 SH, and ethyl, CH 3 .CH 2 SH - and thiolactio acid, CH 3 .CHSH.COOH, have been found as cleavage products on partial oxidation of proteids, though they cannot be regarded as definite nuclei existing preformed in the molecule. In all these compounds the S occurs as E, S - H, and may be split off by boiling with alkalies. Again a certain amount of H 2 S passes off as gas during hydrolysis, and this, since the cystin is not affected, must have been combined in some other form. The proportions of loosely combined and stable S vary much in different proteids, the former being frequently absent alto- gether, whilst some of the albumoses contain only loosely com- bined S. From some of the albumenoids, the keratins particularly, large amounts of cystin, as well as loosely combined sulphur, have been obtained. The close relation of cystin to taurin, a constituent of one of the bile acids, suggests a possible origin for the latter compound. E3H CH,.S0 3 H HE, CH 3 NH 3 H Cy stein. Taurin. The transformation has been accomplished in the laboratory as follows : CH 2 SH CH 2 SO 3 H CHXH, oxidation by Bromine -> CHNH, COOH COOH Cystein. Cystein sulphuric acid. 132 OUTLINES OF PHYSIOLOGICAL CHEMISTEY. On heating cystein sulphuric acid in a sealed tube CO 2 is split off (so called CO 2 condensation), leaving CH 2 S0 3 H CH 2 NH a Taurin. On feeding animals with cystin it is found that the amount of taurin in the bile is markedly increased. Seeing also how easily the change from cystin can be effected artificially it is considered probable that the taurin of the bile is directly de- rived from the cystin of the proteid molecule. Cystin is closely related to serin, a cleavage product of silk fiber. CH 2 OH CHNH 3 COOH a amino /3 oxypropionic acid. Serin. Silk fiber is an albumenoid and therefore a modified albumen. Serin is probably modified cystein. INTERMEDIATE PRODUCTS OF HYDROLYSIS OR PROTEOSES. ALBUMENS MODIFIED BY HYDROLYSIS. Our discussion so far has been confined to the true pro- teids or albumens, and their nuclei, or final products of hydro- lysis. We have already mentioned, however, that there are certain intermediate products, and these may be taken up more in detail. A. ACID AND ALKALI ALBUMENS. On treating albumen with dilute acid or alkali it gradually undergoes a modification which is not very well understood, but in all probability represents the very earliest stages of hy- THE PROTEIDS. 133 drolytic cleavage. H 2 S is given off by alkali, NH 3 by acid treatment ; so there must, at any rate, be a change in the mole- cule of some nature. The albumen in this condition will act as a base in acid solu- tion, combining with the acid to form a salt. In alkaline solu- tion it acts as an acid, forming a salt with an alkali. In either case the salt is soluble, but on neutralizing the solution the albumen is driven from its combination and precipitates out as a coagulum, showing that it is denaturalized. On again acidi- fying or alkalinizing the fluid the albumen again dissolves as a salt. The acid and alkali albumens when in solution are not coag- ulable by heat but can be easily salted out, resembling globu- lins in the latter respect. They are very easily hydrolyzed by boiling with dilute acids or by the action of enzymes. Proteid in this condition may be regarded as intermediate between albumen and the proteoses. Note. It must be understood that this change takes place very slowly in the cold, but more rapidly on warming the solu- tion. In speaking, therefore, of proteids in acid or alkaline solution it does not necessarily mean that the proteid is in the form of acid or alkali albumen. Th( B. PROTEOSES. ie intermediate products which are obtained when any proteid is subjected to hydrolytic cleavage by any method are called proteoses. This is a general term applied to a large number of substances showing considerable differences among themselves, but which still have enough of the proteid character to distinguish them clearly from the final products of hydrol- ysis, i. e., the nuclei. 134 OUTLINES OF PHYSIOLOGICAL CHEMISTEY. The proteoses as a group resemble the true proteids in that they, speaking very generally, give : 1. All or nearly all the color reactions due to definite nuclei. 2. The biuret test, implying that their internal arrangement is similar. 3. The same chemical precipitation reactions with alkaloid reagents or metallic salts. They differ from the true proteids in that they : 1. Do not coagulate by heat or by the action of strong alcohol as a rule. 2. Do not as a rule salt out so readily. 3. Are dialyzable and therefore have a smaller molecule. They are separated from coagulable proteid in solution by boiling and filtering. The filtrate contains the proteoses only. Since their chemical constitutions are not known they can only be grouped according to their physical properties, and their behavior towards ammonium sulphate is the most conve- nient method of differentiating them. I. Albumoses. Precipitated by saturated ammo- nium sulphate. Proteoses < II. Peptones. Not precipitated by ammonium sulphate. I. Albumoses. A great variety of albumoses has been recognized, but we can only attempt to deal with the more important of them. A. Primary. Precipitated by 50 per cent. ammonium sulphate (in acid solution). Albumoses B. Secondary. Precipitated by saturated ammo- nium sulphate. THE PKOTEIDS. 135 Primary albumoses are subdivided into : "1. Heteroalbumose. Precipitated by sat- urated neutral sodium chloride, imary albumoses - 2. Protalbumose. Precipitated by sat- urated acid sodium chloride. 1. Heteroalbumose of all the proteoses is the nearest to true proteids, being the only one which is nondialyzable and pre- cipitable in weak alcohol (32 per cent.). In some respects it resembles the globulins, being insoluble in distilled water, but easily dissolved on adding a little salt. 2. Protalbumose is very soluble in distilled water, and is readily dialyzable, so that its molecular weight must be lower than that of heteroalbumose. It is more soluble in dilute alcohol than in water, but begins to precipitate in 80 per cent, alcohol, although it cannot be com- pletely precipitated except by a mixture of alcohol and ether. The two primary albumoses can be separated by treatment with weak 32 per cent, alcohol. The filtrate contains the protalbumose only. It may be remarked that although alcohol will precipitate the primary albumoses it does not coagulate them. They can always be redissolved and in this respect differ from the true proteids. They are not denaturalized even by long standing under alcohol as is the case with albumens. There is a marked difference in the final cleavage products of the two primary albumoses. Heteroalbumose. Protalbumose. Leucin, little, much. Tyrosin, much, trace. Glycocoll, much, none. Sulphur loosely combined, some, much (1-2%). Neither of them contains a carbohydrate group. 136 OUTLINES OF PHYSIOLOGICAL CHEMISTKY. B. Secondary Albumoses. Three fractions have been distinguished, but it will not be profitable to enter into the details of their differences. More of the true proteid reactions are missing in these than in the primary albumoses, so that in general they represent a further stage of cleavage than the primary albumoses from which they can be obtained by hydrolysis. It is, however, probable, that on hydrolysis of proteid some of the secondary albumoses are split off direct from the proteid molecule without passing through the primary stage. One in particular called synalbumose contains a carbohydrate group which is wanting in the primary albumoses, so cannot have passed through those stages. II. Peptones. The peptones are the last and simplest of the products of hydrolysis, which still retain some of the proteid characteris- tics. They are readily dialyzable and cannot be coagulated by heat or precipitated by alcohol or saturated ammonium sul- phate. On the other hand, they can be precipitated by the alkaloid reagents, showing that they contain diamino acids, and they afford the biuret test, so in their internal construction they must still be closely related to the proteids. They are, therefore, recognized as belonging to the proteids, using the term in its broadest sense. Attempts have been made to classify the peptones according to the ease or difficulty with which they can be hydrolyzed Kuhne's anti and amphopeptone but the distinctions drawn are arbitrary and not of sufficient value to discuss in detail. The little that is known about them indicates that they all closely resemble each other. THE PROTELDS. 137 One point may be noted, that a proteid at first splits into a number of different albumoses, but on further hydrolysis the latter split into peptones between which only slight differences can be detected. Albumen Primary albumose '\ Heteroalbumose Protalbumose Synalbumose Secondary albumoses Peptones Amino acids. III. Binary Compounds. Between the peptones and the final nuclei there are probably some intermediate binary compounds temporarily present, but only one is known, Leucinimid, composed of two molecules of leucin. Arginin and cystin are binary compounds, but they are not split up by hydrolysis. No doubt from the commencement of hydrolysis nuclei are being continually split off as such, and the accompanying table gives a hypothetical summary of the probable course of events during the hydrolysis. The actual number of nuclei in each intermediate product is not known, even approximately, but there are reasons for be- lieving that the nuclei in the original proteid molecule may amount to 100 or 120 in some cases. 138 OUTLINES OF PHYSIOLOGICAL CHEMISTRY. Proteid molecule First Splitting. Synalbumoses. Up to 25 nuclei. Peptone. Up to 10 nuclei. Peptone. Up to 10 nuclei. Third Splitting. r+n + i+i+i+i+i+i Peptone. Up to 10 nuclei. Binary. Compounds. Amino acids. Nuclei. Fourth Splitting. Binary. Compound. Amino acids. Nuclei. up to 120 nuclei. + 1+1+1+1+ Amino acids. Nuclei up to 10. + 1+1+1+ Amino acids. Nuclei up to 5. + 1+1+1+!+ Amino acids. Nuclei up to 15. Amino acids. Nuclei up to 5. Biuret We may compare the table with that drawn up for starch in chapter 7, the processes of hydrolysis in each case being analogous. Polyamino acids. I Amino acids Dextrins Monosaccharides. 1 I I Proteids Albumoses Peptones Polysaccharides I Starch Soluble starch An exact comparison cannot very well be made, since, in some respects, the monosaccharides are more comparable with THE PROTEIDS. 139 the peptones. The monosaccharides are still carbohydrates but the ammo acids are no longer proteid. This is a mere matter of nomenclature but the peptones and monosaccharides are the forms in which the proteids and carbohydrates respectively are absorbed and utilized by the body, so that physiologically they are comparable. Still the amino acids and monosaccharides are the final products of hydrolysis in each case, whilst the peptones and dextrins are intermediate products. By regarding the proteids as polyamino acids and the starches as polysaccharides our method of comparison seems to be justified. ALBUMENOIDS. The albumenoids in their native condition are insoluble but some of them can be hydrolyzed to soluble forms. The final products of their hydrolysis are the same as those of true proteids. Collagen is the chemical basis of white fibrous tissue, forming as such the frame work of the individual organs and tissues, whilst, mixed with lime salts to form the bones, it con- stitutes the framework of the entire body. It is resistant to gastric digestion but can be hydrolyzed by trypsin or boiling with dilute acids. Gelatin is the first intermediate product of its hydrolysis and this stage can be reached by prolonged boiling with water alone. Meat on cooking becomes more tender and this is due to partial hydrolysis of collagen to gelatin. Gelatin itself is not found in the living tissues. Glue, obtained by boiling bones, cartilage, etc., is simply impure gelatin. The various commercial gelatins are more or less purified glue. Gelatin in solution, as is well known, dissolves on heating and solidifies (gelatinizes) in the cold. It is supposed by some that the 140 OUTLINES OF PHYSIOLOGICAL CHEMISTRY. power of gelatinization depends upon the presence of mineral salts, but the purest gelatin obtainable, containing only .04 per cent, of ash, will gelatinize in 1 per cent, solution so that the role of the mineral salts does not appear to be an important one. Gelatin, although tyrosin cannot be obtained from it, gives the Millon faintly and the biuret reaction very strongly so that it is of a proteid nature, but there appears to be some group or groups lacking which are of importance for nutrition since the nitrogen contained in gelatin cannot entirely take the place of proteid nitrogen. Gelatin, therefore, is not a proteid food but it is good sparer of nitrogen. In other words if an absolute gelatin diet is given there is less waste of nitrogen from the tissues than with an absolute carbohydrate or fat diet. Elastin is the basis for elastic fibrous tissue. It is much more resistant than collagen, and requires prolonged action of hydrolytic agents before it is broken down. Both collagen and elastin on hydrolysis afford albumoses and peptones similar to those obtained from ordinary albumen. Of the final products tyrosin can be obtained from elastin though not from collagen. Keratin. The function of keratin is different from that of collagen. It is not a supporting medium but forms the external coverings of the body, horns, hoofs, hair, etc., being secreted by the epithelial cells of the epidermis whilst collagen is secreted by the connective tissue cells. Keratin is the most resistant of all the albumenoids to hydro- lytic agents, and can only be decomposed by boiling with strong acids or alkalis. The main point of interest is its high sulphur content, 4-5 per cent. Cystin is present in large quantities, its chief source for laboratory experiments or other uses being hoofs or hair. THE PROTEIDS. 141 Albumenoids. Digestion with Boiling -with Pepsin. Trypsin. Water. Dilute Acids. Strong Acids. Collagen. Elastin. Keratin. readily slowly to gelatin readily slowly readily readily slowly This merely shows the relative speed of hydrolysis. On boiling with strong acids the intermediate proteoses are quickly decomposed, and the nuclei are decomposed to some ex- tent, so the products cannot be compared with those derived from digestion with trypsin or dilute acids. Cystin, however, is not affected and can be obtained as such from keratin. PROTEIDES. There are certain proteids which by mild action of a hydro- lytic agent may be split into two parts, one of which is always a complete proteid body, the other being organic but not a proteid. This is analogous to the cleavage of the glucosides into two atom groups one of which is always a sugar. Hoppe- Seyler in consideration of the analogy with the glucosides has designated the complex albuminous bodies the proteides. By English authors they are generally called compound proteids. Three classes of these compound proteids may be considered, glyco-proteids, hemoglobins and nucleo-proteids. Albumen 1. Glycoproteids, MueinsS Glycosamin On cleavage with acids these bodies very readily yield a re- ducing substance, glycosamin, and proteid. They are viscous colloid substances of a marked acid nature, dissolving readily in dilute alkalies from which solution they are precipitated by acetic acid. The precipitation by acetic acid is used to obtain the mucin from mixed solutions. 142 OUTLINES OF PHYSIOLOGICAL CHEMISTRY. They are not coagulated by heat but are denaturalized by long-continued action of acids, alkalies or alcohol. Their ele- mentary analysis shows considerable difference from the true proteid, the difference being due to the large amount of glyco- samin, which contains less N and more O than true proteid. c. H. N. s. o. Mucin 48 6.5 11.5 .8 32 per cent. Proteid 52 7 16 .5 22 per cent. Certain mucins have been found to yield as much as 30 per cent, of a reducing substance. The nature of the proteid part of the molecule has been neglected for a study of the reducing substance. MUCOIDS. These compounds have a great similarity to the mucins from which they are distinguished, not sharply, by certain physical properties. They are less slimy than mucins, but the difference between mucins and mucoids are only of degree not of kind. The name mucin is now restricted to the slimy substances secreted by epithelium ; all other compounds having a similar behavior being called mucoids. Mucoids have been obtained from tendon, umbilical cord, white of eggs, blood serum, ascitic fluid and cartilage. They are all easily hydrolyzed with the direct release of glycosamin except in one instance, chondromucoid from cartilage, which has certain characteristic cleavage products distinguishing it from all other mucoids. When chondromucin is boiled with dilute acids there appears, in addition to the usual cleavage products of proteid, a sulphur containing chondroitin. By further cleavage with dilute hydrochloric acid all the sul- phur is split off as sulphuric acid, showing that the original compound was in the form of chondroitin sulphuric acid. Chondroitin has no reducing action, but on further cleavage THE PEOTEIDS. 143 affords a reducing body, chondrosin, and diacetic acid, the latter being at once split up into two molecules of acetic acid. Chondrosin on cleavage with alkali yields glycosamin and glycuronic acid. ^^ mucoid (Acid hydrolysis) Proteid Usual products Chondroitin sulphuric ester Chondroitin Sulphuric acid Chondrosin Diacetic acid (Alkali hydrolysis) / \ Acetic Acetic / \ acid acid Glycosamin Glycuronic acid >. CO-CH 2 CO CH 3 ^H-N=CH (CHOH^ COOH tfHOH), , O SO 3 H Chondroitin sulphuric ester m CO CH 8 CO-CH, CH N=CH (CHOH^ COOH (OHOH), CH 2 OH Chondroitin + sulphuric acid. to H N=CH (CHOH) 4 -COOH (CHOH), Chondrosin Diacetic acid (acetacetic acid). CHO bCH 2 OH jlycosamin (9HOH), COOH Glycuronic acid. 144 OUTLINES OF PHYSIOLOGICAL CHEMISTRY. Chondroitin sulphuric acid therefore is glycosamin with various atom groups attached which can be split off one by one. Phosphoglycoproteids. The mucins and mucoids already considered do not contain phosphorus and in this respect differ from the phospho-glycoproteids. The latter on gastric diges- tion yield a reducing carbohydrate and a phosphorized proteid group. They are of little importance, being only found in invertebrate animals. Globin II. Hemoglobin^ Hematin Hemoglobin (Hb) is the red coloring matter found in the blood of all vertebrates. It consists of a proteid, globin, com- bined with a non-proteid iron-containing pigment. Globin is a member of a peculiar group of .proteids called histons which will be described later. The precipitation and color reactions of hemoglobin are those common to proteids, but it differs in two respects from all other proteids. 1. It crystallizes easily. 2. It has the property of forming loose compounds with oxygen, carbon monoxide, nitric oxide and some other gases. It is present in the red blood corpuscles as a loose compound with some other constituent of the cell. The chief method of distinguishing the varieties of Hb and its derivatives is by means of the spectroscope. Since space does not permit us to deal with spectrum analysis, we can only touch lightly on the hemoglobins. It is necessary to distinguish between oxyhemoglobin (HbO) and reduced hemoglobin (Hb). The blood in the lungs is saturated with oxygen, HbO is formed and in the course of the circulation oxygen is given up again to the tissues, leaving the THE PKOTEIDS. 145 Hb in the reduced condition. The reduced Hb solution is very dark colored (venous blood), whilst HbO is bright red (arterial blood). The amount of oxygen which combines with the Hb is not constant but varies with the pressure of the oxygen gas. In this respect it behaves much as gases in aqueous solution. By exhausting blood under the bell jar of an air pump all the oxygen may be removed. HbO crystallizes more easily than Hb, and is therefore more readily obtained in pure form. Being thus by far the most easily crystallizable of all proteids it has been the favorite one used in molecular weight determinations. The empirical for- mula has been approximately determined as C 758 H 1203 N 195 O 218 FeS 3 for dog's hemoglobin. METHEMOGLOBIN. In addition to the labile oxyhemoglobin, there is another oxygen compound of hemoglobin. This, methemoglobin, is stable in that it will not give up its oxygen in a vacuum. A great many substances have the power of inducing the forma- tion of this compound, and it is often formed in the blood ves- sels by the action of poisons. In this condition the oxygen cannot be utilized by the body cells. CARBON MONOXIDE HEMOGLOBIN, HbCO Is formed when carbon monoxide comes in contact with hem- oglobin. It is a comparatively stable substance ; the gas is given off in a vacuum very slowly. If the carbon monoxide is present in the air it combines with the hemoglobin to the exclusion of the oxygen because it has a greater affinity for and forms a more stable compound with it. The tissues die for lack of oxygen. The great danger in gas poisoning at the 10 146 OUTLINES OF PHYSIOLOGICAL CHEMISTKY. present day is due to the fact that a large percentage of the gas furnished is carbon monoxide. Hemoglobin forms compounds with many other gases, but they are not of sufficient importance to describe here. HEMATIN. Hematin is the non-proteid iron-containing pigment obtained by cleavage of hemoglobin, a change which takes place very quickly in acid solution. If oxygen is excluded during the cleavage hemochromogen and not hematin results. Hemo- chromogen may be considered as reduced hematin, the former being a ferro compound while the latter isferri. The oxidation and reduction of this substance is accomplished almost as read- ily as with hemoglobin, and there are reasons for believing that the latter is an ester of the acid hematin with the basic globin. Hematin itself does not crystallize, but chlorhematin obtained by the action of HC1, by which Cl is substituted for an OH group, crystallizes readily and has been the subject of much study. C 32 H 32 N 4 Fe0 4 + HC1 m+~ C 32 H 31 ClN 4 FeO 3 + H 2 O Hematin. Hemin or chlorhematin. The hemin crystals obtained from different animal species are to some extent characteristic. Hematin on cleavage with acids, e. g. HBr, yields hemato- porphyrin, a pigment containing no iron, which has been shown to be isomeric with one of the bile pigments, bilirubin. Hema- toporphyrin is much darker than hematin. The bright red color of the latter is due to the Fe. 2H 2 O + 2HBr = 2C 16 H 18 N 2 3 -f FeBr 2 + H a Hematin. Hematoporphyrin. It is of interest to note that chlorophyll, the iron-containing respiratory pigments of plants, yields a similar compound, phyl- THE PROTEIDS. 147 loporphyrin, on cleavage with acids. The other bile pigments are derived from bilirubin by oxidation and reduction processes. III. NUCLEO-PROTEIDS. Nucleo-proteids are the chief constituent of cell nuclei and are therefore most abundant in glandular organs. They are compounds of proteid with nucleic acid, and the generally ac- cepted view of their decomposition is as follows. Nucleo-proteid Nuclein Proteid r \ Nucleic Proteid acid /n Pyrimidin Purin Phosphoric Bases. Bases. acid. All uucleo proteids do not pass through the exact number of steps given above, and this scheme applies only in a general way. The proteid part of the molecule, as well as the nucleic acid, shows great variation. But a characteristic of these com- pounds is the high percentage of phosphorus. C. H. N. P. S. O. 48.5 7 17 1.5^ .7 24. When subjected to gastric (pepsin + .2 per cent. HC1) diges- tion, proteid is split off and hydrolyzed to soluble albumoses and peptones, but the residue, (nuclein), containing all the phos- phorus, remains undissolved. Nuclein, therefore, is much richer in P than the original nncleo -proteid, containing 3-5 per cent. Gastric juice has little effect upon nuclein, but in the in- testine the digestion is completed. 148 OUTLINES OF PHYSIOLOGICAL CHEMISTRY. All the P of the original molecule is found in the nucleic acids of which the elementary percentages are : C 37ft H 4ft N 15ft O 34ft P 10ft Nucleic acid can be dissolved in strong acids, and if to such a solution albumose is added, precipitates are formed which show a remarkable similarity to nucleo-proteids, indicating that proteid, or its modifications, has a strong affinity for nucleic acid. On further digestion with trypsin or on boiling with strong (5 per cent. HC1) acid, nucleic acid is broken up into its final products, phosphoric acid and the soluble purin bases. The nucleo-proteids differ to some extent according to the different organs from which they are obtained and some of them yield pyrimidin bases also, thymin, uracil, but these are of much less importance than the purin bases and need not be considered. The different nucleic acids yield varying propor- tions of the following : Adenin C 5 H 5 N 5 Guanin C 6 H 6 N 6 O also called Xanthin bases or Hypoxanthin C 5 H 4 N 4 O Alloxuric bases. Xanthin C 5 H 4 lSr 4 O 2 The simplest method of obtaining the purin bases from the products of proteolysis is by precipitating them with silver nitrate, AgNO 3 , from the solution made strongly alkaline with ammonia. AgNO 3 + NH 3 + H - purin base = NH 4 NO 3 + Ag - purin base. The silver compound thus formed is decomposed by H 2 S, forming insoluble AgS, and setting the bases free. Uric acid, C 5 H 4 N 4 O 3 , trioxypurin, is an oxidation product of these bases and it has been found that in the course of THE PROTEIDS. 149 metabolism in the human body a portion of the nuclein waste is excreted as uric acid. Such a decomposition as that figured above for nucleo-proteids, resulting in purin bases, takes place in the body tissues, and it has been proved that certain organs, liver and thymus, can oxidize a portion of these to uric acid. The method of demon- strating this is of interest. The liver is taken from the animal at the moment of death, ground to pulp as quickly as possible, nuclein or purin bases added, and the whole kept at 40 C. In three or four hours uric acid is found to be present. It seems probable that all tissues take part in such a formation of uric acid in proportion to their content of nuclein. The nuclein bases taken in the food suffer the same fate as those originating from destruction of body tissue and accordingly the total uric acid excreted may be divided into two protocols. The Endogenous, that part which comes from the purin bases of body tissue. The Exogenous, that portion which comes from the purin bases of the food. In digestion the nucleo-proteids are split up by pepsin in the stomach to proteid + nuclein. The nuclein is then decomposed by trypsin to nucleic acid -f- proteid in the small intestine, and the nucleic acid to the purin bases which are absorbed as such ; little or none passing out with the feces. With an ordinary meat diet the purin bases absorbed are in excess of the needs of the organism, the excess representing the exogenous portion of the uric acid. When the diet is regulated so that no purin bases are taken in with the food the uric acid output is materially lessened. Much of the experimental work on the production and destruc- tion of uric acid in the animal body has been confusing because different species of animals used by different investigators rueta- 150 OUTLINES OF PHYSIOLOGICAL CHEMISTRY. bolize proteid in dissimilar ways. Obviously it is impossible to gain exact knowledge of uric acid metabolism in man by a study of birds because the latter excrete practically all of their waste nitrogen as uric acid, which calls for a synthetic formation of the same whilst in man and other mammals such a process is probably of little importance. It has been stated above that uric acid may be formed in the liver by simple oxidation of xanthin or hypoxanthin but this statement tells only half the truth, for the process continues and results in a destruction of a portion, at least, of the uric acid thus formed. There are thus two processes taking place simultaneously in this organ and in one animal species the formation, whilst in another the destruction of uric acid is the predominant action. For instance, if a dog's liver is removed immediately after the death of the animal, tubes inserted in the hepatic artery and vein so that defibrinated blood may be passed through and the whole kept at a temperature of 38 C. the organ is surviving and shows its normal activities. By carefully oxygenating the blood supply the organ may be kept alive for some hours. Such perfusion experiments have proved that uric acid added to the arterial blood is oxidized (to urea) by the liver cells. The cells of other tissues, muscles, kidneys, etc., do the same, so that not all the uric acid formed in the course of metabolism is excreted as such and in a general way we may say that the uric acid excreted represents the portion which has escaped oxidation in the body. Besides the breaking down there is constant building up by cells to repair waste. Cell metabolism maintains the necessary equilibrium. We may therefore trace three sources for urea, two of which have been dealt with previously. THE PROTEIDS. 151 1. Glvcocoll, obtained by oxidation of the ordinary proteid nuclei. 2. Guanidin hydrolyzed from its combination with ornithin. 3. Uric acid derived from nucleic acid. We may again point out that uric acid is not always a pre- cursor of urea as was formerly supposed. Nucleo Proteids In tissues (Cell metabolism) In food (Gastric digestion) Nuclein Proteid (Tryptic digestion) Proteid Nuclein ./\ Proteid Nucleic acid / Nucleic acid Proteid (Tryptic digestion) Phosphoric acid Purin bases Purin bases Phosphoric acid (Oxidized by (Absorbed and liver, &c. ) oxidized by liver, &c. ) f Uric acid Endogenous (Partly oxidized by liver, &c.) Exogenous. Urea, CO 2 , H 2 O (Excreted). Uric acid (Excreted). 152 OUTLINES OF PHYSIOLOGICAL CHEMISTRY. OTHER PROTEIDS. We have so far considered typical proteids, the albumens, and their modifications, but there are some proteids adapted for special purposes, which have not the character of albumens. They appear to be closely related to the albumens but are not modifications of them in the sense in which proteoses and albumenoids are regarded as modifications, so will be discussed as separate groups. One can think of them as specialized albumens. I. Phosphorized Proteid. Casein, the principal milk proteid, is the most important rep- resentative of this group. Casein does not coagulate on boiling but its nutritive proper- ties are impaired, and it is found that some sulphur is split off, so it must undergo some chemical changes. In addition, 1. It contains no carbohydrate group. 2. It contains phosphorus. 1. Is of physiological interest. Free sugar exists in milk in the form of lactose, and it is from this that the sucking mammal derives its carbohydrates, so there is no necessity for these being present in the proteid source of nourishment. On the other hand eggs contain no free sugar and the embryo bird can only derive its carbohydrates from the carbohydrate group combined in the egg albumen. 2. Since casein contains phosphorus it resembles the nucleo- proteids in this respect and is often called a pseudo-nuclein or nucleo-albumen. But nucleic acid is absent, so these terms are misleading and it is better to regard casein simply as a phos- phorized proteid. As with the nucleo-proteids the P is present as phosphoric acid in combination, but it is here in direct com- bination with the proteid, whilst in the nucleo-proteids the P THE PROTEIDS. 153 is a constituent of the nucleic acid, not being found in the split- off proteid. Casein. Nucleo-proteid. Proteid phosphoric acid. Proteid nucleic acid. Purin Phosphoric bases. acid. Phosphorus is necessary for the suckling and is obviously supplied by means of the casein. In the egg nucleo-proteids are present in the yolk, and afford the phosphorus required by the embryo bird. Whilst on the subject of phosphorus a slight digression may be made. Phosphorus is required not only for building up the nucleo-proteids of the nuclei, but also for nervous tissue which contains a large amount of P in the form of lecithin, a phosphorized fat occurring in combination with proteid. II. Basic Proteids. Histons and protamins. These differ from albumens : 1. In not being coagulableby heat. 2. In being distinctly basic. They have no acid qualities, so do not give the amphoteric reaction of the albumens. This basic character is due to the relatively large quantities of the hexon bases they contain. They afford most of the color reactions, but contain no car- bohydrate group. The biuret reaction is very marked and this is probably due to the comparatively large number of free NH 2 groups. (a) The histons on account of their distinctly basic qualities combine more readily with acids than the ordinary albumens, and are therefore specially adapted for the combinations occur- ring in some of the proteides. 154 OUTLINES OF PHYSIOLOGICAL CHEMISTRY. 1. Hemoglobin is a liiston in combination with the acid hematin. 2. Some of the nucleo-proteids are histon in combination with nucleic acid. These are the two forms in which histon occurs in animal tissue, and in studying histon the acids must first be split off in either case. But any method which can be employed for this purpose must at the same time bring about a partial hydrol- ysis of the proteid, so that the histons as obtained for study cannot be regarded as the exact representatives of the original proteid part of the molecule. For instance, histons are not coagulated by heat but hemoglobin and the histon containing nucleo-proteids are coagulated. In these cases it cannot be the hematin and nucleic acid respectively which are responsible for the coagu- lation. The only way of distinguishing the proteid part (histon) of these substances from true albumens is by noting its distinctly basic qualities, and observing that on hydrolysis it yields the hexon bases in larger quantities than usual. There is no reason for supposing that the average histon molecule is smaller than that of the albumens, although they are often assumed to be simpler substances. This is no doubt true of the form in which they are studied, but probably not of that in which they exist in the entire molecule. In speaking of the proteid of hemoglobin and of the nucleo- proteids as albumens in the earlier part of this chapter this was done for the sake of simplicity and in order to avoid con- fusion. It is now evident however that albumen and histon , are so closely allied that it is difficult to say there is any real difference between them. Histon probably does not vary from true albumens more than albumens may vary among them- THE PROTEIDS. 155 selves. It only varies in a direction which makes a distinction easier, i. e., in being decidedly basic. (5) Protamins vary more from the albumens than the histons, being even more strongly basic, and possessing a simpler structure. Most of our knowledge concerning them we owe to Kossel, who considers them as representing the central portion, or proteid nucleus, around which the more complicated albu- mens are built up. But there is no conclusive evidence to show that this is the case. They are never found as intermediate products of hydrolysis, but exist preformed in fresh tissues from which they can be obtained direct. The protamins are not widely distributed, their principal source being the spermatozoa of fish. It is likely that they are specially adapted for fertilizing processes, but this has not been demonstrated. The chief feature of the protamins is the large amount of arginin they contain, sometimes over 80 per cent., e. g., sper- matozoa of salmon (salmin). Since the protamins are nondia- lyzable the molecule must be large, and it is quite possible that their molecular weight is not much below that of albumen, but being built up of fewer kinds of nuclei, their structure may be considered simpler in this respect. Applying the symbol used for the proteid molecule, we may suppose that of each ten nuclei in salmin protein, eight are ar- ginin. Arginin. Arginin. Leucin. Arginin. Arginin. We may suppose the linkage to occur as usual by means of the alpha-ammo acid groups, so that for each additional mole- cule of arginin there would be an increase in basic qualities due to the free guanidin end of the chain. The protamin may be considered as polyarginin to the extent of 80 per cent., whilst 156 OUTLINES OF PHYSIOLOGICAL CHEMISTRY. the average albumen is polyleucin to the extent of 40 per cent, or 50 per cent. To separate protamins from other proteids the solution is boiled. This disposes of the coagulable proteids, and the pro- tamins can be separated from other non-coagulable proteid by NH 3 . They exist as soluble salts in combination with an acid, and the NH 3 drives them from combination, forming NH 4 salts. The protamins thus liberated are insoluble and precipitate out. Unlike the histons, the protamins can be obtained in their native condition. No hydrolytic action is necessary to drive them from a special combination. The protamins on hydrolysis yield intermediate products called protons. These appear to be somewhat different from the proteoses, but the differences are probably simply due to the relatively large quantities of arginin. A table of hexon bases found in various proteids may be given for comparison. Arginin. Lysin. Histidin. Salmin. Protamin from salmon sperm. Sturin. Protamin from sturgeon sperm. Histon from thymus. Gelatin. Casein. Egg albumen. 84 58 14.5 9 4.5 0.8 12 7.5 5 2 trace 12 1.2 trace 2.5 III. Melanins. Pigments. The group tryptophan or proteinochromogen which has recently been identified as skatolamino-acetic acid, has long been considered to be the precursor of animal pigments. Melanoidin is the term applied to pigments obtained on artificial hydrolysis with acids whilst melanin applies to the natural pigments of the skin, hair and retina. They are both very resistant bodies and in origin probably have no relation to THE PROTEIDS. 157 the blood coloring substance hematin. The high sulphur con- tent of many of them does not accord with such an origin, nor do the greater part of them contain any iron. Mdanoidins. The structure of those coloring substances which are obtained by boiling proteids with mineral acids is very complex and most of the work done thus far has been in the line of ultimate analysis. Such work has not been without some results, for it has established that these " melanoidins " as they are called have wide variations in composition. But the one important fact in regard to structure is that on fusion with caustic potash they yield indol and skatol which harmonizes with the tryptophan idea of origin. Melanins. The difficulty of getting the melanins in pure form has hindered a study of their structure but so far as this has been possible, elementary analysis shows marked differences in composition. Fusion with potash yields indol and skatol bodies as with the melanoidins, but until more complete studies are made of their decomposition products it is of little use to say more about them. On fusion with potash indol and skatol may be formed by condensation of tyrosin as well as from tryptophan and some pigments appear to be directly derived from tyrosin by a proc- ess of oxidation. The liquid resembling ink found in the squids has been shown to be such an oxidation product of tyrosin. Since the melanins are absolutely resistant to hydrolysis, it is impossible, as with albumens or protamins, to find out of what nuclei they are composed. About all we know is that fusion of a very minute amount with KOH gives rise to very bad smells, of skatol and indol, so that in all probability the tryptophan or tyrosin rings or both are very largely repre- sented. Taking them at about 70 per cent., and using our 158 OUTLINES OF PHYSIOLOGICAL CHEMISTRY. symbol for the last time we may suppose the melanins to be constructed somewhat as follows : Tryptophan. Tyrosin. Leucin. Tyrosin. Tryptophan. Glutamic acid. With the ink of squids and some vegetable pigments the entire molecule appears to be constructed of tyrosin or some oxidized form of tyrosin so that these may be considered as polytyrosins. Our contention that these groups of proteids just discussed are specialized albumens seems to be justified. 1. Casein is especially adapted for sucklings in containing phosphorus and in being easily digested. Since no carbohy- drate group is required, this is dispensed with. 2. Basic Group. (a) Histons. Specially adapted for combination with cer- tain acid radicals which are required for definite purposes. (5) ProtammSj probably specially adapted for fertilization. 3. Melanins. Resistant bodies specially adapted for absorp- tion of heat and light rays. This property of the melanins cannot be discussed here, since it entails the question of spec- trum analysis. We have said there are reasons for supposing that the proteid molecule may be composed of 100 to 120 nuclei. To close the chapter we may explain those reasons briefly. The molecular weight of proteid is difficult to estimate for various reasons, not the least important being the difficulty of getting a proteid in pure condition. This however does not apply so forcibly to hemoglobin which is easily crystallizable. Its molecular weight has been esti- mated in various ways. 1. By absorption, 100 grams Hb absorbs .2246 grams O 2 . .2246 : 100 : : 32 : 1^250 molecular weight. THE PROTEIDS. 159 2. Sulphur content is .43 per cent. With lead acetate only part of the S is parted with, so there must at any rate be two atoms of S in the molecule. S 2 = 64. .43 : 100 :: 64 : 14,883 molecular weight. /Hematin 4.2 per cent, molecular weight is 592. 3. Hb \Globin. 4.2 : 100 :: 592 : 14,100 molecular weight. 4. Estimation from Fe content works out at about 16,000. These estimations agree fairly closely, so we may take the average at 15,000. Deducting 600 for hematin leaves 14,400 for the molecular weight of the globin. The average molecular weight of the nuclei is about 140, and each time a nucleus enters into combination there is loss of H 2 O, having a molecular weight of 18. We may then take the average nucleus in combination as 120 molecular weight. 14,400 -r- 120 = 120 ; about the probable number of nuclei in the proteid molecule in this instance at any rate. TABLE OF PKOTEIDS. 1. Albumen, 2. Albumen modified by digestion. xAlbumoses Proteoses^ ^Peptones 3. Albumen modified by body cells. /Collagen Albumenoids^- Elastin \Keratin 4. Albumen plus something else. a. Albumen + glycosamin = Glycoproteids. b. u (histon) -f- hematin = Hemoglobin. c. " (sometimes histon) -f nucleic acid = Nucleo-proteids. 5. Albumen specialized. a. Albumen phosphorized, Casein. b. Albumen basic. Histon and protamin. c. Albumen aromatized. Melanins. CHAPTER VII. ENZYMES. A WORK on physiological chemistry no matter how elemen- tary cannot be considered complete at the present time without a few words on the subject of Enzymes. Enzymes are soluble unorganized ferments secreted by cells, both animal and vegetable ; their main object being so to pre- pare the food for the cells that the latter can readily assimilate it. The action occurs quite independently of the life of the secreting cell : it is not a vital process. Until recently a distinction was always made between organ- ized and unorganized ferments ; the latter being the enzymes under consideration, and the former various micro-organisms bacteria, yeasts and moulds which were supposed to exert their ferment action solely by means of vital proc- esses. This, however, is a distinction without a difference, since it is now known for certain that micro-organisms also effect ferment actions by means of soluble enzymes which they secrete just as is the case with cells of the higher animals and plants. The term " vital process " may be objected to as being merely a cloak to cover our ignorance. That is precisely what it is. We can follow up the processes which go on in indi- vidual cells or the body as a whole, and explain them as due to purely chemical or physical reactions up to a certain point, but beyond this we know practically nothing. Our knowledge of what is meant by life is a little more ex- tensive than it was, but even yet is a mere nothing by compari- 160 ENZYMES. 161 son with our ignorance. The cloak covers a little less than it did, but it is the same old cloak still. It has hitherto been found impossible to isolate enzymes and study them in a pure condition, so that their constitution is un- known, and it is even uncertain if they are of a proteid nature or not, but their functions have been very thoroughly studied and a large number of different kinds are known. 1. Enzymes are catalyzers. An astonishingly small amount of an enzyme can apparently exert its action upon an unlimited amount of a given substance, and emerge with undiminished vigor. It is not used up during the reaction. Again the enzymes are simply accelerators of reactions which would ultimately take place without their aid. They do not originate the reaction in which they are concerned (p. 10). 2. The action of enzymes is specific. A given enzyme will act on a given substance or group of closely allied substances, and on no other. This rule however is not universal ; several exceptions are known. SUSCEPTIBILITY. Enzymes are very susceptible to certain external influences. Heat above 65 C. to 70 C. and certain poisons will destroy them, although some substances which are poisonous for a cell are not so for its enzymes. Chloroform, thymol, toluol for instance will destroy the life of a cell but have little or no effect on enzymes. Mercuric chloride is a poison for both. Chloroform or thymol are often added to a solution in which the action of enzymes is being studied. They prevent bacterial growth which might vitiate the results, but do not affect the enzymes. 11 162 OUTLINES OF PHYSIOLOGICAL CHEMISTRY. NOMENCLATURE. The suffix " ase " is given to an enzyme and this is preceded by the name, or its root, of the substance upon which it acts. Thus the enzyme which acts upon Maltose is called Maltase. This momenclature, however, has not yet been universally adopted and it is not possible to use it in every instance. ZYMOGENS OR PROFERMENTS. Enzymes, more particularly those of the digestive tract in animals, often exist in the cells in an inert form, and only be- come active at the moment of their discharge from the cell. In this form they are called zymogens or pro-enzymes. Zymogens may develop into the active state even after the death of the cell which contains them. If a pancreas, for example, is minced fine immediately after death and the juices pressed out, the extract will digest fibrin and other proteids very slightly, but after keeping the pancreas for some days aseptically extracts from it will digest fibrin very readily. The digestive enzyme was present in the first instance as trypsinogen, which on keeping changes to the active trypsin. The enzymes secreted by animals, plants and the lower in- termediate forms of life, are in the main similar, although there are some substances peculiar to plants, such as glucosides, which are acted upon by plant enzymes alone. It is naturally the animal enzymes which most interest us. Having thus given briefly a few points, some of which will be discussed more in detail as occasion arises, about enzymes as a whole, we may proceed to classify them according to their functions. 1. The hydrolyzing enzymes, which may be called Hydrases. 2. The coagulating (clotting) enzymes, which may be called Coagulases. ENZYMES. 163 3. The oxidizing enzymes, which may be called Oxidases. Another classification may be made. 1. Those which act extracellularly, i. e., are diffused out of e cell to do their work, into the intestinal canal, for instance. 2. Those which act intracellularly, i. e., are retained within cell and work there. These two groups, however, are alike in their action, i. e. y an zyme acting on starch or glycogen will affect it in precisely the same way whether it is an extra or intracellular one, so that the difference is only one of location. We will, therefore, dis- cuss the individual enzymes according to the former grouping. I. THE HYDKOLYZING ENZYMES OR HYDBASES. The substances of most interest to us which can be hydro- lyzed by enzymes are carbohydrates, fats and proteids. A. Enzymes Hydrolyzing Carbohydrates. 1. Enzyme hydrolzing starch (Polysaceharide). Amylase (amylum starch) splits the complex starch molecule into maltose ; a disaccharide, with dextrin as an intermediate product. Starch as it occurs in seeds, roots, etc., is insoluble and the first action of the enzyme is to split up the highly com- plex insoluble starch molecule into a less complex form of starch which is soluble. The soluble starch is then simplified to dextrins and these in turn to maltose. But it is probable that during the whole process maltose is being continually split off as such, so that the process may be put diagrammatically as : 12 monosaccharides. 10 monosaccharides. Starch (insol.) = Maltose + Soluble starch. + I II I + I Soluble Starch. Maltose. Dextrin. Dextrin. and the dextrin then hydrolyzed to maltose. 164 OUTLINES OF PHYSIOLOGICAL CHEMISTRY. The reserve material stored up in seeds and bulbs consists principally of starch, and as these begin to grow the starch is converted by amylase secreted in special cells into maltose in which form it can be utilized by the newly forming sprouts. Vegetables taken as food by animals contain large quantities of starch which is attacked by the extracellular amylase of the saliva (ptyalin) and pancreas (amylopsin) and so converted into the soluble disaccharide maltose. Glycogen or animal starch is stored up in the liver and can also be hydrolyzed to maltose by amylase. Amylase is present in the liver cells and here acts as an intracellular enzyme. 2. Enzymes Hydrolyzing Disaccharides. Disaccharides cannot be assimilated as such, but must be first hydrolyzed to monosaccharides. This is effected by means of enzymes of which the principal are : (a) Saccharase hydrolyzes saccharose to glucose + fructose. (Invertase.) (Cane sugar.) (Invert sugar.) (6) Maltose hydrolyzes maltose to glucose -f glucose. (c) Lactose " lactose to glucose + galactose. (Milk sugar.) (a) Saccharase or invertase is secreted by the cells of the small intestine and the products of its action on saccharose glucose and fructose can then be absorbed and assimilated. (b) Maltose is also secreted in the small intestine, where it splits up the maltose already prepared by amylase (ptyalin and amylopsin) into two molecules of glucose. Maltase also prob- ably exists in the blood, since maltose which finds its way there in small amounts is changed to glucose. Large amounts of maltose introduced into the circulation appear in the urine unchanged. (c) Lactose is apparently not secreted in the intestine or elsewhere in the body, but a great deal of lactose is taken in by sucklings with the mother's milk. It seems probable that ENZYMES. 165 the lactose contained in milk is split up in the intestine by bacteria and thus becomes available for assimilation. The Bacillus coli communis, which is a normal inhabitant of the large intestine, secretes lactase : Many varieties of coli communis also secrete saccharase and it is possible that a part of the saccharose ingested is rendered available by this means. B. Enzymes Hydrolyzing Fats. Lipase (XtVo? fat) is secreted by the pancreas (steapsin) and is discharged into the duodenum, where it splits the fats into glycerin and a fatty acid. The fats are emulsified by the juices in the intestine and be- ing thus in a very fine state of division are easily reached by the lipase. The glycerin is absorbed as such, and the fatty acid, combin- ing with any sodium or potassium present, forms a soluble soap. It is in the double form of glycerin and a soluble soap that the fats reach the cells ; once there the fat is built up again. Lipase is also found in most of the body cells as an intracellular enzyme. C. Enzymes Hydrolyzing Proteids. Proteolytic Enzymes Proteases. The proteases break up the complex insoluble, or if soluble non-dialyzable, proteid molecules to peptones which can be absorbed by the intestinal cells, and to some extent the peptones still further to amino acids. Pepsin, secreted in the stomach, and trypsin in the pancreas, are always so fully treated of in works on physiology that they need not be discussed here in detail. Two additional animal proteolytic enzymes have recently been described. Erepsin and Enterokinase. Erepsin is said to be secreted by the cells of the small in- testine, its function being to extend the action of trypsin, tryp- 166 OUTLINES OF PHYSIOLOGICAL CHEMISTRY. sin according to this idea being unable to carry digestion farther than peptones, the erepsin then hydrolyzing the peptones to amino acids. Enterokinase, secreted by the cells of the intestine is said to activate trypsin, the latter being of itself quite inert at the moment of passing into the intestine. If a pancreatic fistula is made in a dog the juice passing out can be collected. Such juice is said to have no digestive action on fibrin or other proteids, but it is at once activated on adding a little juice from the small intestine. The latter is said to con- tain the activating enterokinase. It may be that the trypsin is present in the form of a soluble zymogen. Statements about these new enzymes however must still be accepted with some reserve. The Clotting Enzymes. Coagulases. Rennet which clots milk and the fibrin ferment (thrombase) which clots blood, like pepsin and trypsin, are always so fully discussed that they need not be more than mentioned here. The presence of calcium salts seems to be necessary for their action and it is thought that clotting is due to the formation of a calcium salt of the casein or fibrin as the case may be. But this is still uncertain ; the exact way in which the coagulases act is not known. Plasmase is a recent discovery. It is secreted in the stomach and is said to have the property of coagulating albumen and albumoses which are then digested by the proteolytic enzymes. But this requires confirmation. Oxidizing Enzymes. Oxidases. It has always been a puzzle why certain substances such as albumen, which are oxidized with great difficulty in the labo- ratory, can be easily oxidized in the body, whilst on the other ENZYMES. 167 hand certain substances, such as oxalic acid, easily oxidized by the chemist, pass through the body unchanged. Until recently it was necessary to put these oxidation processes under the heading of " vital processes," but the exist- ence of specific oxidizing enzymes in plants is now proved and it has been sought to show that such occur also in animal tissues : specific enzymes which will oxidize definite substances but allow others to pass by. The oxidizing enzymes are carriers of oxygen. It is sup- posed that they yield oxygen to the substances to be oxidized and then instantaneously reoxidize themselves. In this they differ from oxidizing substances such as potassium permanganate and chromic acid, which, once deprived of their O, remain reduced and inert. Some Fe and Mn oxides and salts, how- ever, can yield O and then reconstruct themselves, but their action is slow compared with the oxidizing enzymes which work with inconceivable rapidity. Oxidases have the power of turning guaiac tincture blue, by oxidizing it, and are divided into two main groups according to the differences in their action on it. 1. Those which turn guaiac blue direct, utilizing the O = O of the air for their reconstruction : Direct Oxidases. 2. Those which do not turn guaiac blue until the addition of H 2 O 2 . Indirect or Peroxidases. All protoplasm contains an enzyme, Catalase y which has the power of reducing H 2 O 2 to H 2 O and O. The liberated O = is then used by the peroxidases, but they cannot use O = O of the air to reoxidize themselves. The O must be nascent. Briefly. \. Direct Oxidases utilize O = O. 2. Peroxidases utilize O = . 168 OUTLINES OF PHYSIOLOGICAL CHEMISTRY. Plants contain both of the oxidases, but in animal tissues only the latter are present. Animal peroxidases are never poured out into the digestive tract ; their field of action is altogether intracellular. Animal tissues then will liberate O = from H 2 O 2 by means of their catalase, and the question then arises : " Is not the oxidation of the guaiac due simply to the O = liberated by catalytic action on H 2 O 2 ?" That it is not so oxidized but requires a carrier can be shown by means of bacterial cul- tures. Take an agar culture of any common bacillus and pour into the tube 2 or 3 c.c. of an emulsion of gum guaiac, and then 1 or 2 c.c. of H 2 O 2 . There is very active evolution of gas. O= must be given off in great abundance, yet there is no bluing of the guaiac. In 10 or 15 minutes add a little blood. A blue color immediately appears. The bacteria contain catalase but no peroxidase. We may take it as probable then that catalase and peroxidase are two separate enzymes ; the one reducing and the other transferring the O = to other substances. It seems legitimate to suppose that the reduction of oxyhem- oglobin (HBO) in the body is due to the catalase which thus becomes the medium by which O == is supplied to the tissues ; the peroxidase acting as carrier and so assisting oxidation. But we must be very careful not to attach much importance to peroxidase. We know that fats, sugars and proteids are oxi- dized in the body, and we know that, outside the body at any rate, these are not influenced by peroxidase in the least. All we know about peroxidase is that it will oxidize guaiac, but we have no idea what else it can do. It has been claimed that apart from the peroxidases enzymes are present in the liver and some other organs, which even ENZYMES. 169 after death will oxidize aldehydes (e. g., salicyl aldehyde) to acids. These have been called aldehydases. But aldehydes are so greedy for oxygen that the mere pres- ence of O= is sufficient for their oxidation, so it appears super- fluous to assume a special oxidizing enzyme for this. O = lib- erated by catalase is all that is needed for oxidation of aldehydes. The guaiac test for oxidases is simple and may be briefly described. Put some lumps of gum guaiac into a test-tube containing alcohol and boil over burner until deep yellow. Filter and add the filtrate to water in a test-tube or porcelain dish to the point of a milky emulsion. 1. Into this put a slice of potato. A deep blue color quickly appears which diffuses around the slice. Direct ox- idases. 2. Instead of potato put in some blood or a piece of animal tissue minced fine. No reaction. Add a little H 2 O 2 and a blue color quickly appears. Peroxi- This is a classical test for blood. Even if the blood has been dried out for months it will give the reaction. 3. Bubbles appear on the surface of the tissue. Catalase. The H 2 O 2 is being reduced to H 2 O and O. If the potato or animal tissue is previously boiled no reac- tion occurs. Both the catalase and the oxidases have been de- stroyed. As has already been said, of the peroxidases we know prac- tically nothing beyond the fact that they exist, but of the direct plant oxidases there are several different kinds which afford various definite reactions, but the classification into two main groups as already given is as much as can be attempted here. 170 OUTLINES OF PHYSIOLOGICAL CHEMISTRY. We may however mention tyrosinase. Certain fungi (Russula and Boletus), if cut with a knife, turn red or blue and finally black on the cut surface. The coloring is due to a melanin formed by the action of a direct oxidizing enzyme, tyrosinase on tyrosin. Both tyrosin and tyrosinase ex- ist free in the plant but only come in contact when the tissues are injured. The tyrosin is oxidized by the enzyme, and the oxidation products condense into a highly complex melanin. Whether the enzyme causes the condensation as well as the oxidation is not known. The ink of the squid to which we have already referred is a similar substance also formed by tyrosinase, and it may be that some other animal pigments are produced in the same way, but it is not yet known if this is the case or not. Tyrosinase extracted from russula or boletus is often used as a test for tyrosin. The merest trace of tyrosin in a solution can be detected in this way by darkening of the fluid. REVERSING ACTION OF ENZYMES. Referring again to the hydrolyzing enzymes we found that they break down complex into simpler substances without be- ing themselves affected. They are catalyzers. But although the enzymes are not used up, yet they are never able entirely to complete the change. Taking maltase as an example : If this is added to concen- trated, 20 per cent., solution of maltose, it will change 86 per cent, into glucose, but leave 14 per cent, unchanged. The per- centage varies with the dilution. In a dilute solution a larger percentage is changed but there is always some maltose left. It was formerly thought that the glucose formed acted as a re- strainer of the enzyme action but this is not the case, for on ENZYMES. 171 taking a solution in which the action has ceased, and adding more maltose the action begins again, although there is just as much glucose present as before, and this renewed action does not cease until a definite relation is attained between the amount of maltose and glucose. There is a point of equilibrium (p. 7) beyond which the enzyme is unable to carry the action. Within the last few years a very curious fact has been as- certained with regard to maltase. If it is added to a concen- trated, 40 per cent, solution of pure glucose it reverses its action, and starts in to build up maltose. It will do this until the nat- ural equilibrium for this particular concentration is reached, i. e. 9 14 per cent, of maltose. Then the action stops. There is com- plete analogy with purely chemical reactions. Constant break- ing down of maltose but constant building up at the same rate. Glucose 86 14 "Maltose E. Point of equilibrium. Such a reversing action has been demonstrated for maltase and lipase, but not so far for other enzymes, although it is quite possible they may have the same power. If this is proved to be so it will be a decided step in advance, since it will show that not only breaking down but also building up can be carried on apart from the actual life of the cell. But there are still innumerable reactions taking place in the cell to account for which no enzymes have yet been discovered. We are for instance quite ignorant to what extent the per- oxidases are responsible for oxidation processes. Even if enzymes are discovered for all these reactions there still remains the question : " How are enzymes formed ? " 172 OUTLINES OF PHYSIOLOGICAL CHEMISTRY. If enzymes are constructive as well as destructive the ob- vious uses of the intracellular enzymes are enormously extended. They are no longer mere scavengers, intended to break down and clear away what is no longer needed by the cell, but are the agents by which the cell maintains the necessary equilibrium between its protoplasmic contents and those of the surrounding medium, i. e., the extracellular blood or lymph serum. PLANT ENZYMES. Among the plants all the enzymes found in animal tissue are represented, with the exception of the fibrin ferment (Throm- base), but on the other hand there are a number of enzymes peculiar to the vegetable kingdom. Among the more important are : 1. Cytase (cellulase), which hydrolyzes cellulose to sugars. 2. Inulase, which hydrolyzes inulin (a polysaccharide allied to starch) to sugar. 3. Glucosidases, usually called glucoside splitters, which hydrolyze glucosides to glucose and something else (p. 91). There are a large number of different glucosides in plants and a large number of different enzymes which split them. One glucoside and its enzyme may be referred to. Plant indican is a glucoside and the enzyme splitting it is called indicase. Its action is as follows : /N 1 glucose -, OH I N/ I S J +H 2 = I I 7 I + glucose NH NH Plant indican. Indoxyl. We may recall urine indican. Urine indican. Indoxyl. ENZYMES. 173 The indoxyl in each case then oxidizes itself by means of Q = O as already described (p. 92) to indigo blue. Indigo is obtained by crushing the leaves of the indigo plant. Indoxyl is liberated by the indicase and changes to indigo blue. The chlorophyll is then dissolved out of the leaves by alcohol in which indigo blue is insoluble, and the indigo blue then dis- solved out in chloroform to separate it from the cellulose skele- ton of the leaf. What the uses of glucosides are to a plant are somewhat obscure. The glucoside and its enzyme exist side by side in the cell, of a leaf for instance, and it is not until the leaf is bruised or dies that they come in contact and the reaction occurs. Since many of the products of the splitting are dis- agreeable to the taste it has been thought that these may serve as a warning to an animal which is eating a leaf, not to eat another, so that the storing up of the glucoside and its enzyme may be considered as a protective measure. But in some cases the products are not disagreeable, to our taste at least, and certainly herbivorous animals will eat many glucoside-containing leaves with apparent satisfaction. It may be that, in some instances at any rate, this is a convenient way of storing glucose so that it is ready for an emergency. The leaf is injured, glucose is liberated by the enzyme, and the leaf uses it for building itself up again. The glucose is anchored to a cyclic compound to preserve it in stable form until needed. AUTOLYSIS. If a piece of animal tissue or organ is taken from the body aseptically and kept free from bacteria, decomposition, as it is ordinarily understood, does not take place. If such aseptic tissue is heated up to 70 C. or 80 C. for an hour or so, it remains unchanged indefinitely, but otherwise it 174 OUTLINES OF PHYSIOLOGICAL CHEMISTRY. undergoes changes which are not those of decomposition (bac- terial action) but analogous to the digestive processes which go on in the intestinal tract. It is gradually digested by the proteolytic enzymes contained in the cells. Autodigestion (Autolysis). The products are the same in both instances, albumoses, pep- tones, and amino acids. Hydrolysis takes place but no oxida- tion. The peroxidases, by means of which oxidation may be partly carried on, are there, but are powerless to act because only O = O is offered them instead of their accustomed O = derived from HbO. The cells of some organs also contain amylase and lipase, so that on autolysis the glycogen and fats if present are hydrolyzed to glucose and fatty acids. CHAPTER VIII. DISEASE AND IMMUNITY. ALTHOUGH the subjects treated of in this chapter belong to pathology rather than physiology the two are so closely allied jfchat a brief sketch may be attempted. In a general way we may say, Disease is caused by absorption of poison. Immunity is defense against poison. 1. Poisons may be mineral or organic. The organic poisons may be subdivided into : 1. Those secreted by plants, e. g., \egetable alkaloids. 2. Those secreted by animals, e. g., snake poisons. 3. Those secreted by intermediate forms of life, e. g., bac- terial poisons. 4. Waste or split products of any of these three forms of life, which may or may not be poisonous. These may be called accidental poisons. They are not formed by the organism with a view to defense or attack. PTOMAINS. TOXINS. LEUCOMAINS. It is necessary to have a clear understanding of what is meant by these terms. A. PTOMAINS. Bacteria may be divided into two groups ; the pathogenic, which can multiply in the body and cause disease, and the saprophytic, which are not able to multiply in the living body. Decomposition of flesh is due to saprophytic bacteria and in the process they split up the proteid matter and furnish certain 175 176 OUTLINES OF PHYSIOLOGICAL CHEMISTRY. soluble basic products, chiefly amins and oxyamins, called Ptomains (TTT^GL corpse). The ptomains are not necessarily poisonous but they may be and the danger of eating decompos- ing meat or fish is due to their possible presence. The ptomains belong to group 4. B. TOXINS. Toxins are synthetic products of bacteria. They are not the split products of proteids but are sub- stances synthesized by the bacteria inside the bacterial cell. When formed they may diffuse out of the cell or be retained within it. As an example of each case we may take the bacilli of diphtheria and typhoid. If a culture of diphtheria bacilli in broth is grown for two or three weeks and then filtered through a porcelain filter, the filtrate, although free from bacteria, is poisonous. It contains a soluble toxin excreted by the bacilli. If a culture of typhoid bacilli in broth is grown for two or three weeks and then filtered through a porcelain filter, the filtrate is harmless. It contains no toxin. But if the bacilli remaining on the filter are dried, thoroughly pulverized and suspended in water, the suspension is poisonous. The bacterial cells contain an intracellular toxin. The courses of the two diseases also illustrate this. The diphtheria bacillus does not penetrate the tissues, but grows locally and superficially, forming a membrane on the surface of the throat for instance. But from this point its soluble toxins are absorbed into the system and cause the symptoms of the disease. Toxemia. Typhoid is a slow lingering disease. The bacilli get into the circulation and grow there. Period of invasion. After a time some begin to die off and, the bacteria disintegrating, the intra- DISEASE AND IMMUNITY. 177 cellular toxins are liberated, and cause the fever. Later the bacilli gradually disappear from the blood, but the fever con- tinues as long as any are left. Bacteremia or Septicemia. C. LEUCOMAINS. Leucomains are the products of the splitting of proteid by body cells. Leucomains are analogous to ptomains and may be identical. For instance if lecithin is acted upon by bacteria, cholin may be split off. Cholin in this case is a ptomain. If lecithin is acted upon by a body cell, cholin may be split off. Cholin in this case is a leucomain. Leucomains however do not leave the body as such in health. They undergo oxidation to urea, CO 2 and H 2 O. But in certain pathological conditions, they may not be properly oxidized, and remaining in the system, act as poisons. Autointoxication, uremia for instance. IMMUNITY. Immunity is the resistance of the body to poisons and its capability of ridding itself of toxic agents such as bacteria. Immunity may be natural or acquired. It takes a certain amount of any given poison to kill, and the smallest amount that will effect this is usually called the minimum lethal dose, M.L.D. Supposing a rabbit can stand an injection of .09 c.c. of diphtheria toxin, but that .1 c.c. will kill it, .1 c.c. is the M.L.D. and the rabbit has a natural im- munity against .09 c.c. A few days after a first injection of .09 c.c. into a rabbit a second dose may be given, and it now takes a much larger amount to kill; perhaps .2 c.c. By carefully raising the amounts the rabbit will soon be able to stand, say 1.0 c.c. or \ ten times the M.L.D., without being affected. 12 178 OUTLINES OF PHYSIOLOGICAL CHEMISTRY. To its natural immunity against .09 c.c. an acquired immunity against .9 c.c. has been added. It is now immune to 1 c.c. or ten M.L.D. and is said to have been immunized to diphtheria toxin. Antitoxins. If some of the serum of this rabbit is injected into a second rabbit and at the same time a dose of diphtheria toxin, greater, even considerably greater, than the M.L.D. the second rabbit does not die. The serum of the first rabbit contains something Anti- toxin which protects the second rabbit. The first rabbit has been actively immunized with toxin. The second rabbit has been passively immunized with anti- toxin. This is the basis on which the antitoxin treatment of diph- theria has been built up. Just what antitoxin is, is still a matter of doubt, but since experiments have shown that toxin can be neutralized by anti- toxin " in vitro," i. e., in test-tubes in the laboratory, the neu- tralization clearly cannot be due to " vital action." Again the antitoxin is used up in the process so it is not a catalyzer, and its action is not analogous to that of enzymes. There must, therefore, be a purely chemical combination be- tween the toxin and antitoxin, and, taking this for granted, Ehrlich has constructed a theory which, for the present, serves very well as a working hypothesis. EHRLICH'S THEORY OF ANTITOXINS. Every cell is built up of an innumerable number of proteid molecules, which can be divided into two groups. 1. The central molecules, which are concerned in cell metab- olism. 2. The peripheral molecules, which are concerned in cell nu- trition. DISEASE AXD IMMUNITY. 179 DIAGRAM L . The peripheral molecules are built up of a large number of central nuclei, amino acids, etc., in combination, and possess in addition side chains which are chemically active. We may consider benzoic acid C 6 H 5 .COOH as analogous, the COOH being a chemically active side chain. By means of their side chains the peripheral molecules are able to combine with side chains of various molecules passing along in the blood current and anchor them. Analogy. Benzoic acid anchors glycocoll to form hippuric acid. Such anchored mole- cules may be passed on to the central molecules and utilized by them. A toxin molecule is anchored in the same way, but instead of being an assistance it paralyzes the side chain and throws it out of action. If many of the side chains are thus thrown out of action the cell dies, and if a suffi- cient number of cells are thrown out of action, the entire organism dies. The toxin has killed the animal. But the amount of toxin anchored may only be sufficient to injure the cell temporarily, so that it can recover. It is a well-known fact that injury is followed by hypertrophy, i. e., over- production of the tissues locally at the point of injury. For Example. A callus is formed as a broken leg heals. Just so with the injured cells. There is over-production at the site of injury, i. e., the side chains of the A *> Antitoxin occupied by toxin. peripheral molecules (usually called cell receptors). These M t Molecules of cell ; 3/ u Mole- cules with receptors ; R, Recep- tor; Jg lf Receptor with toxin ; T, 180 OUTLINES OF PHYSIOLOGICAL CHEMISTRY. may be produced in great excess, and a number of them be- come detached and float away in the blood current. These cast-off side chains or receptors are the antitoxins. They have now acquired specific properties, i. e., will an- chor the particular toxin which caused their production but nothing else. Additional amounts of this particular toxin now introduced into the circulation, do not reach the cells, but are anchored and rendered inert by the antitoxins in the blood current. The cells, therefore, escape injury. (Diagram 1.) The success of the antitoxin treatment of diphtheria led to hopes being entertained that all bacterial diseases could be equally well overcome by means of antitoxins. This, however, is not the case. Tetanus and diphtheria kill by toxemia and the body cells form antitoxins to resist this, but most pathogenic bacteria cause disease and death by bacteremia and the immunization process against this is somewhat differ- ent. Antitoxins are not formed in bacteremia or only to a limited extent ; not sufficient for the blood to protect other ani- mals passively. IMMUNIZATION AGAINST BACTEREMIA. The typhoid bacillus can be taken again as an example. AGGLUTININS. The blood serum of a patient with typhoid fever acquires the property of agglutinating typhoid bacilli. Observed in a hanging drop under the microscope the bacilli are seen to be very motile. If a drop of diluted normal blood serum is added, the motility is not affected, but on adding a drop of diluted serum of a typhoid patient the bacilli lose their motility, and collect together in clumps. DISEASE AND IMMUNITY. 181 The clumping can also be observed macroscopically in a test-tube. If to 1 c.c. of a typhoid culture in broth 1 c.c. of typhoid serum, diluted say to 1-25 with physiological salt solu- tion, is added, making the total dilution 1-50, the mixture soon appears flaky, small clumps become visible which gradually sink to the bottom of the test-tube, leaving the fluid above perfectly clear. This is called the clump or Widal reaction and is very gen- erally employed for diagnosis of typhoid fever. The serum of rabbits or other animals inoculated with cul- tures of typhoid bacilli also acquires agglutinating properties. By repeated injections the serum can be made very active, so that dilutions of 1-20,000 or even higher may show the reaction. It appears that under the in- fluence of the immune serum a sticky substance is exuded from the bacilli. This is called the agglutinum ; and the active substance in the serum, the agglutinm. The agglutination, however, does not kill the bacilli. It only makes them somewhat inert, so that in the presence of agglutinins they cannot mul- tiply or only very slowly. Ehrlich's theory with regard to this is that some of the side chains of the peripheral mole- cules of a body cell are sup- plied with two groups, a haptophore (seizing), and a toxophore (toxic) group. (Diagram II.) DIAGRAM II. AP o Bacillus H, Molecules of cell ; M lt Molecules of Bacillus; AP, Agglutinophore Group; H t Haptophore Group ; R, Receptors occupied by agglutinins; A, Free Agglutinin; A lt Agglutinin with Receptor. 182 OUTLINES OF PHYSIOLOGICAL CHEMISTRY. The haptophore group H seizes a receptor K of the bacillus and the toxophore or agglutinophore group AP influences the bacillus in such a way that it exudes the agglutinum. If the body is invaded by the bacilli so many of these parti- cular side chains are thrown out of action that the cell is injured to this extent. There is therefore over-production, and many of the new side chains are cast off into the circulation where they occur as free agglutinins. The agglutinins are specific. They will agglutinate the bacterium of invasion but no other. PEECIPITINS. A similar curious phenomenon has also been observed. If a broth culture of typhoid three or four weeks old is filtered through porcelain and thus freed from bacilli, the clear filtrate, if immune serum is added, will shortly become flaky, with formation of clumps which gradually sink to the bottom of the test-tube. This is the precipitin reaction. The active substance in the serum is called the precipitin and the passive substance in the filtrate which is precipitated is called the preeipitum. The serum in this case must be much more concentrated than for agglutination. A serum which will agglutinate typhoid bacilli in dilution of 1-10,000 will probably not cause precipi- tation in a filtrate in higher dilutions than 1-20 or 1-40. Filtrates of fresh cultures cannot be precipitated in this way. The preeipitum is probably an albumenous substance liberated on the death of the bacilli, and is no doubt closely allied to the agglutinum though it is still an open question if the two are identical or not. LYSINS. The agglutinins, as already mentioned, do not kill but only hold the bacteria in check, yet the bacteria must be killed in order to get rid of them. DISEASE AND IMMUNITY. 183 Normal serum if fresh will kill a few bacteria, and this power is greatly increased against any particular bacterium on immuni- zation with it. This increased power can be more clearly demonstrated " in vivo " than " in vitro/' by means of what is known as PfeifFer's phenomenon. Pfeiffer experimented with cholera bacilli but the same thing applies to typhoid. If a large number of typhoid bacilli are injected into the peritoneal cavity of a normal guinea-pig (or rabbit, etc.), and a small quantity of the peritoneal fluid withdrawn from time to time by means of a capillary glass tube, microscopical examina- tion shows that the bacilli after two or three hours are still numerous and actively motile. There are more of them than the normal fluid can kill and in all probability they will multiply and set up a fatal bacteremia. If the same thing is done with a guinea-pig immunized to typhoid the bacilli immediately lose their motility and clump. Before long they become granular and begin to break up. Cul- tures taken from time to time show that the bacilli are killed within a few hours. The agents which kill them are called bacteriolysins. An analogous case is that of the hemolysins, i. e., agents which destroy red blood corpuscles, and since the action of these is much more easily demonstrated " in vitro " than the action of bacteriolysins it is chiefly on experiments with hemolysins that Ehrlich has built up his theory of the lysins. We can take two animals, the goat and the rabbit, as examples, but what applies to them applies to other animals also. Normal rabbit serum can dissolve the red corpuscles of a goat to a very limited extent in a test-tube, but this power is enhanced to a remarkable degree if the rabbit is treated with successive injections of goat's blood. The serum of the rabbit has become strongly hemolytic for goat's corpuscles. 184 OUTLINES OF PHYSIOLOGICAL, CHEMISTRY. As an example of an experiment we may dilute 1 c.c. of hemolytic serum with 99 c.c. physiological salt solution ; take 1 c.c. of the dilution in a test-tube and into this put one drop of defibrinated goat's blood. As the red corpuscles dissolve the hemoglobin diffuses out and the fluid becomes red. To see if there has been total hemolysis or not a microscopical exami- nation can be made. If such hemolytic serum is warmed to 60 C. for 30 minutes it will no longer destroy goat corpuscles. It has become inactivated. Supposing we now take normal rabbit serum and add a few drops of it to the fluid. The red cells are quickly dissolved. The inactivated serum has been reactivated by normal serum. Why does normal serum have this power? We shall see directly. With bacteriolysins there is no simple method of observa- tion like this, as bacteria have to be added to the serum, a loopful of the fluid plated out at intervals and the colonies counted ; a tedious and not very accurate method. But the bacteriolysins act in just the same way as hemolysins, so we can now return to our typhoid bacilli to work out the meaning of these phenomena. Ehrlich's theory extended to the lysins, supposes that some of the peripheral molecules of the body cells contain side chains specially constructed to grapple with bacteria, and probably other inert foreign bodies and destroy them. It must be remembered that the bacteria themselves appear to the body cell simply as inert foreign substances. It is only their toxins which actually cause direct injury. Such special side chains contain two groups, a chemically active haptophore group, and a latent receptor group. The haptophore group can seize a receptor of a bacillus and DISEASE AND IMMUNITY. 185 anchor it, but cannot of itself exert any influence on the bacillus. But as soon as it has anchored the bacillus its latent receptor group becomes chemically active and capable of receiving a body which is normally present in the blood called a complement (also called alexine or addiment). The combined action of the complement and special side chain can destroy the bacillus. But although the cell is not directly injured these special side chains are thrown out of action, and consequently overproduc- tion takes place : Many are cast off and circulate free in the blood current. They are now known as " immune " or " inter- mediate 9 ' bodies or ambocep- tors, and with the aid of the com- plements destroy the bacilli. The intermediate bodies like the antitoxins and agglutinins are produced in excess in im- munization, but the comple- ments do not increase in num- ber. They exist normally in the body and the cells, i. e., the leucocytes, which produce them are not in any way affected by the bacteria so there is no tendency to over-production. Complements are destroyed by heating to 60 C. and are said to be thermolabile, whilst the intermediate bodies are only affected at considerably higher temperatures, 70-7 5 C., so are called thermostable. We can now understand why immune serum is inactivated Bacillus M, Molecules of cell ; M lt Molecules of ba- cillus ; It, Receptors : H, Haptophore Group; J, Free intermediate body ; J,, With com- plement and receptor ; C, Free complement ; C lt With receptor ; (7 2 , With intermediate body. 186 OUTLINES OF PHYSIOLOGICAL CHEMISTRY. by heating to 60 C. (Complements are destroyed, but not intermediate bodies), and can be reactivated by adding normal serum. (Intermediate bodies supplied with fresh complements.) Again complements once separated from the body quickly break up and disappear from the serum, whereas intermediate bodies remain intact for months. For this reason serum used in experiments with lysins must be fresh, not more than one or two days old. But as with heated serum old inactive immune serum can be reactivated with fresh normal serum. Pfeiffer's phenomenon does not show with a normal animal because there are only complements present in the peritoneal fluid, whilst with an immunized animal both bodies exist free in the peritoneal cavity. The intermediate bodies are specific. They will only attack the particular species of bacterium or blood cell against which the animal has been immunized, whilst the complements are general, being able to combine with any intermediate body, although it may be that bacterial complements are not the same as hemolytic complements. It must be confessed that this has been put in a very general way. There are numerous partial exceptions to the rules here laid down and numbers of cases which do not seem to conform to them at all. The consequence of this is that Ehrlich has been obliged so to extend and complicate his theory to meet all the requirements, that it is becoming doubtful if it will stand the strain much longer. Some new and more simple theory for the phenomena of bacteremia is urgently needed but as yet there is none in sight, and for the present we must make the best we can of Ehrlich's. A study of the reactions against bacteremia and the theories to account for them makes one curious to know what is the meaning of the double and apparently independent action of the DISEASE AND IMMUNITY. 187 agglutinins and bacteriolysins. We can only guess, but it seems quite possible that agglutination is a means of holding the bac- teria in check until they can be destroyed. On introducing bacteria into the circulation some are at once destroyed by the cell side chains and complements, and as soon as intermediate bodies are formed still more can be destroyed. But the complements are soon used up and it is necessary to wait for a fresh supply. Meanwhile side by side with the inter- mediate bodies free agglutinins have been produced and these help to prevent multiplication until more complements are ready. The question then arises : " Why is not some provision made for a larger supply of complements so that the bacteria may be destroyed more quickly ? " The answer seems to -be, that it would not do to kill the bacteria off too quickly. It must be remembered that they contain intracellular toxins which are only set free on the death of the bacilli and against these in- tracellular toxins antitoxins do not appear to be formed or at any rate only to a limited extent, so that the liberation of large amounts of toxins all at once would be dangerous and liable to cause the death of the organism. True in Pfeiffer's phenomenon a large number of bacteria can certainly be killed in a short time, yet there is always a limit to the number, depending on the number of complements present. We are dealing here too with highly immunized animals which can no doubt stand a large dose of toxin even though there may be little or no antitoxin in the blood. It is quite possible that, instead of the organism as a whole becoming immunized to the intracellular toxin by means of antitoxin in the circulation, the individual cells gradually get hardened to it. Analogous cases can be found in mineral poisons. An arsenic (88 OUTLINES OF PHYSIOLOGICAL CHEMISTRY. eater can take enough arsenic in 24 hours to kill a dozen ordi- nary people, yet there are no antiarsenic bodies in his blood. One can only suppose that the individual cells have accustomed themselves to the poison in some way, so that they are no longer injured. It may be said that no antibodies have ever been experi- mentally produced in serums against poisons of a relatively simple nature whose chemical constitutions are known. In order to avoid misunderstanding it may be mentioned that a number of workers claim to have succeeded in obtaining anti- typhoid serums, the use of which is beneficial in cases of typhoid fever. But these serums have never come into general use and the tendency is to discredit them. The same may be said of antistreptococcus and antipneumococcus serums. On the other hand recent experiments by Yaughan indicate that the toxins of typhoid and intracellular toxins in general are relatively simple bodies, against which one would not ex- pect antitoxins to be raised. By breaking down the bacterial cells with sulphuric acid he has extracted virulent toxins which resist boiling. Highly complex molecules would certainly be destroyed under these conditions. Before closing the chapter a further reference to the pre- cipitins must be made. It appears that any albumenous substance when injected into an animal gives rise to precipitins in the serum, i. e., "anti- bodies " which will precipitate it from solution, and moreover the anti-body is always specific for that particular albumen. In this way a large number of albumens which had always been supposed to be precisely alike have been shown to differ to some infinitesimal extent, a difference which cannot be detected by their ordinary chemical or physical (e. g., salting out) re- actions. DISEASE AND IMMUNITY. 189 Differentiation of albumens by this means is called the bio- logical test. Examples: 1. If the milk of a cow is injected into a rabbit, the serum of the latter will precipitate the casein of cow's milk, but not that of human or other milk, or only to a very slight extent. 2. If the blood of a cow or ox (beef blood) is injected into a rabbit the serum of the latter will not only contain hemolysins but also precipitins, so that on adding a few drops of the immune rabbit serum to 1 c.c. clear beef serum, the latter becomes flaky with precipitation of clumps to the bottom of the test-tube. This reaction is made use of as a test for human blood. The serum of a rabbit immunized to human blood (human- ized rabbit) will cause a precipitate in diluted human serum or even in an aqueous solution of blood which has been dried out for months, so that by this means it can be determined if blood stains are those of an animal or human being. The precipitin is specific though not absolutely so. A humanized serum which will precipitate human serum at 1-100 may precipitate that of an ape at 1-30, and that of a dog at 1-10 for example. The more nearly related the animal the more likely is its blood to react. There is probably strict specificity for the par- ticular albumen, but other kinds of blood may contain small proportions of it. We may suppose that human blood contains of albumen A 50 per cent, and of B 50 per cent., both of which give rise to precipitins. The blood of an ape may have 10 per cent, of A, 20 per cent, of B and 70 per cent, of C, whilst that of a dog has 10 per cent, of A, 10 per cent, of C and 80 per cent, of D. If three rabbits are immunized, each with one kind of blood 190 OUTLINES OF PHYSIOLOGICAL CHEMISTRY. to such an extent that the serum of each reacts at 1-100 with its blood of immunization we would have : Human Serum. Ape Serum. Dog Serum. Kabbit No. 1 with human blood 1-100 1-30 1-10 " " 2 " ape " 1-30 1-100 1-20 " " 3 " dog 1-10 1-20 1-100 We have throughout said that the agglutinins and lysins are specific, but they are not absolutely so any more than the pre- cipitins. It has therefore been suggested that all these reac- tions should be called special rather than specific, but there seems no good reason for this. They are specific enough for all practical purposes so may just as well be designated as such. Probably all anti-bodies, not only precipitins, but also lysins and agglutinins are formed, not against particular cells, but against particular albumens contained in those cells. Thus a rabbit serum which will agglutinate the typhoid bacillus 1-20,000 may agglutinate the Bacillus coli communis 1-100 or 1-200. This simply means that Bacillus coli communis contains a small proportion of the albumens which go to make up the protoplasm of the typhoid bacillus, Immunity. Against albumens. Against simpler substances. Antibodies formed and No antibodies. Probably exist free in individual cells accus- serum. torn themselves to the poisons. ~| Against toxic Against inert albumens albumens. acting as foreign bodies. Antitoxins. Agglutinins. Precipitins. Lysins. POSTSCRIPT. 191 POSTSCRIPT. While this book was going through the press a new formula for tryptophan was suggested by Ellinger which explains very well the relations existing between this substance and kynurenic acid. . CH a NH, /\ CH COOH CH Tryptophan. Kynurenic acid. When tryptophan is fed to a dog kynurenic acid is ex- creted. If gelatin, which does not give the Adamkiewicz reaction and therefore does not contain the tryptophan group, is fed kynurenic acid is not formed. The new formula for tryptophan is probably the correct one but it does not affect the comparison made between the products of tyrosin and tryptophan on p. 128. INDEX. Acids, 30 Adamkievicz reaction, 94 Agglutinins, 181 Albumens, 100 Albumen acid, 132 alkali, 132 Albumoses, 136 Alcohols, 26 Aldehydes, 28 Alkaloid reagents, 117 Alkaloids, 95 Allotropism, 15 Amid N, 61 Amids, 59 Amino acids, 64 diacids, 69 Amins, 62 Amylase, 163 Anhydrids, 38 Antitoxins, 178 Arginin, 121 Asymmetrical C atom, 40 Autointoxication, 177 AutolysiS) 174 B Bacteremia, 180 Benzene ring, 74 excretion, 81 Binary compounds, 137 Biological test, 188 Biuret, 60 Butane, 24 Cadaverin, 108 Carbohydrates, 47 Carbon molecule, 35 Carbonic acid, 38 Carbylamin, 63 Casein, 152 Catalase, 167 13 Catalysis, 10 Cholesterin, 90 Cholin, 71 Chondroitin, 143 Coagulases, 166 Coagulation, 101 Collagen, 139 Colloidal sol., 11 Complements, 185 Cresols, 83 Cystin, 131 D Diacids, 39 Diamino acids, 70 Diatomic alcohols, 39 Diffusion, 11 Dioxybenzenes, 78 Disaccharides, 50 Dissociation, 1 acids, 4 bases, 4 Double rings, 90 E Elastin, 140 Enterpkinase, 166 Equilibrium, 7 Erepsin, 165 Esters, 37 Ethane, 24 Ethers, 36 F Fats, 44 Fehling'ssol., 33 Formulas, empirical, 16 graphic, 18 Furfurol, 91 a Gelatin, 140 Glucosides, 91 Glycocoll, 65 Glycoproteids, 141 193 194 INDEX. Glycoflamin, 70 Glycuronic acid, 48 Guaiac test, 169 Guanidin, 61 H Hematin, 146 Hematoporphyrin, 146 Hemin, 146 Hemoglobin, 144 Hexon bases, 123 Hippuric acid, 82 Histon, 153 Homologues, 25 Hopkin's reaction, 94 Hydrocarbons, 24 Hydrolysis, 52 Hydroxylamin, 85 Immunity, 177 Indican plant, 172 urine, 92 Indicanuria, 93 Indigo blue, 92 Indol, 92 Intermediate bodies, 185 Ions, 2 Isomers, 25 K Keratin, 140 Ketones, 28 Lactic acid, 40 Lecithin, 71 Leucin, 65 Leucomains, 177 Lipase, 165 Lysin, 122 Lysins, 182 M Melanins, 156 Melanoidins, 156 Mercaptans, 54 Methane, 23 Millon's reaction, 77 Molisch' s reaction, 90 Monpsaccharides, 48 Mucin, 141 Mucoids, 142 Mustard oils, 63 N Naphthalene, 90 Nitrils, 56 Nitrobenzene, 76 Nitrosoindol, 94 Nitroso-reaction, 68 Nitrous acid action, 67 Nucleic acid, 147 Nuclein, 147 Nucleo-proteids, 147 Oils, 46 Ornithin, 70 Osazones, 89 Osmosis, 15 Oxidases, 166 Oxidation, 13 Oxyacids, 40 Oxybenzenes, 75 Peptones, 136 Peroxidases, 167 Pfeiffer's phenomenon, 183 Phenol, 75 ^ Phenylalanin, 83 Phenyl hydrazin, 84 Phenylisocyanate, 84 Piria's test, 125 Polysaccharides, 50 Precipitation, 101 Precipitins, 182 Propane, 24 Protamins, 155 Proteases, 165 Proteids, alkaloid tests, 117 bases, 123 color tests, 116 construction, 110 nitrogen, 117 , nuclei, 106, 107 phosphorized, 152 phosphorus in, 153 products of bacteria, 108 body cells, 109 hydrolysis, 106 oxidation, 108 sulphur in, 130 Proteides, 141 Proteoses, 133 INDEX. 195 Ptomams, 175 Purin bases, 95 Putrescin, 108 Pyrimidin bases, 98 Quinone, 79 Reactions, 3 Reduction, 13 Reversion, 7 enzymes, 170 S Salting out, 101 Salicylic acid, 82 Saponification, 52 Serin, 132 Skatol, 93 Soaps, 45 Substitution, 23 Sulphur compounds, 54 Synalbumose, 136 Tartaric acid, 43 Taurin, 132 Toxaemia, 176 Toxins, 176 Triatomic alcohol, 39 Trioxybenzenes, 80 Tryptophan, 93 Tyrosin, 83 condensation, 127 Unsaturated compounds, 34 Urea, 60 production, 97 Uric acid, 95 production, 149 Wax, 46 Xanthin, 95 Xanthoproteic reaction, 78 Zymogens, 162 THIS BOOK IS DUE ON THE LAST DATE STAMPED BELOW AN INITIAL FINE OF 25 CENTS WILL BE ASSESSED FOR FAILURE TO RETURN THIS BOOK ON THE DATE DUE. 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