EXCHANGE An Experimental Study of Certain Basic Ammo Acids DISSERTATION SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIRE- MENT FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN THE FACULTY OF PURE SCIENCE, -: COLUMBIA UNIVERSITY BY MARY L. CALDWELL, A.B., A.M. New York City 1921 The Jackson Prcs, 1921 An Experimental Study of Certain Basic Ammo Acids DISSERTATION SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIRE- MENT FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN THE FACULTY OF PURE SCIENCE, COLUMBIA UNIVERSITY BY MARY L. CALDWELL, A.B., A.M. New York City 1921 The Jacksoa Press, Kingston 1921 ACKNOWLEDGMENTS This investigation was undertaken at the suggestion of Professor H. C. Sherman, and was carried out under his direction. The author wishes to express to Professor Sherman her appreciation of his advice and encouragement received throughout this work. The author also wishes to thank Professor A. W. Thomas for many helpful suggestions. M. L. C. AN EXPERIMENTAL STUDY OF CERTAIN BASIC AMINO ACIDS The object of this investigation was to study the influence of certain basic amino acids upon the hydrolysis of "soluble" starch by purified pancreatic amylase. And since, at the time the inves- tigation was started, it was impossible to buy arginine and his- tidine, it was decided: First, to prepare them in a form suitable for the enzyme work ; second, to study their action with the enzyme. PREPARATION OF ARGININE AND HISTIDINE The preparations of arginine and histidine were obtained from commercial gelatin and casein by the method first worked out by Kossel and Kutscher 1 in 1900-1901. This method is based on the separation of histidine and arginine from the other amino acids present in the hydrolysis mixture by means of their silver salts, and then from each other by the difference in solubility of these salts in neutral and in strongly alkaline solutions. This method has since been modified and improved by several workers Kossel and Patten, 2 , Steudel, 3 Kossel and Pringle, 4 Weiss, 5 Osborne, Leaven- worth and Brautlecht, 6 and has been summarized by Plimmer. 7 In carrying out the preparation it was found that this method, with its modifications, presented difficulties; being vague and inde- finite in places, and conflicting directions being given. An attempt was therefore made to standardize the process more fully. The preparation was as follows : Hydrolysis and Estimation of Protein. About 100 grams of protein were hydrolyzed by boiling with a mixture of 300 grams of concentrated sulfuric acid and 600 grams of water under a reflux condenser for 24 hours in an oil bath which was kept at about 105 C. A biuret test at this point was always negative, indicating complete hydrolysis of protein to amino acids. The solution thus iZ. physiol. Chem., (1900-1901) 31, 165-214. 2 Ibid (1903) 38, 39-45. sibid (1902-1903) 37, 219-220; (1905) 44, 157-158. *Ibid (1906) 49, 301-321. 'Ibid (1907) 52, 107. Am. Jour. Physiol. (1908) 23, 180-200. 7 The Chemical Constitution of the Proteins, Part I (1917), 55-63. 502089 obtained was then filtered, if not clear, and made up to 1 liter with water. Nitrogen determinations were made in 5 cc. portions by the Kjeldahl method, and from these determinations the total amount of protein in the mixture was calculated, since the per- centage of nitrogen in the pure protein was known. Removal of Sulfuric acid. Most of the sulfuric acid was re- moved from the solution by adding a little less than the calculated amount of barium hydroxide. This was added in the form of a hot solution, slowly, with constant stirring, until the reaction of the main solution was alkaline to Congo-red, but still acid to litmus. 1 At this point the recommendations of previous investigators were conflicting, and sometimes vague. The use of barium hy- droxide, in the form of a hot or a cold solution, or in the form of a solid, has been advocated, and it was to be added "until the re- action is only faintly acid and almost the whole of the sulfuric acid is precipitated as barium sulfate." The use of the hot solution was found best. The precipitate formed is easier to filter, and the danger of decomposing the ar- ginine present by local excess of alkali is decreased. The use of Congo red paper was adopted as indicating the correct concentration of hydrogen-ion at this point. The precipitate of barium sulfate thus formed was carefully washed by stirring up with water in a mortar and filtering by suc- tion. This was repeated until the filtrate gave no precipitate with a solution of phosphotungstic acid. Usually three washings were sufficient. Evaporation. The filtrate and washings were evaporated ir> vacuo at 70 C. and made up to 1 liter. A determination of nitrogen was made with 5 cc. portions of this solution. This gave, by dif- ference, the amount of nitrogen removed in the first barium sulfate precipitate.. This leads to another inconsistent point in the methods as pre- viously described. It is recommended that the above evaporation of the slightly acid solution be carried out in vacuo, while later on in the processes, alkaline solutions are evaporated in open evapo- rating dishes at 100 C. This is the case in the removal of ammonia J Dakin: J. Biol. Chem. (1920) 44, 499-529. from the solution made alkaline with barium carbonate, and also in the Kossel and Pringle modification for separating the histidine silver compound from the arginine silver compound, by warming and then boiling the solution with barium carbonate. Since it had been found best to carry out some of the evapo- rations in vacuo, it was decided that it would be more consistent to carry out all evaporations in vacuo, at 70 C. This decision was found to be justified by experiment, the difference being especially noticeable in the yield of arginine, as would be expected. In two experiments with casein, one conducted partly in vacuo and partly in open evaporating dishes, and the other in vacuo entirely, the yields of nitrogen in the arginine fractions were 0.35 per cent, and 1.1 per cent of the protein respectively. In the vacuum distillations three-liter flasks were found con- venient, as the larger flasks made evaporation more rapid, and less- ened the danger of loss from foaming. An ordinary water-pump was used, and a pressure of 30 mm. or less was maintained. Oil baths were found to be better than water baths, as the temperature remained more constant, and trouble by evaporation of the water was eliminated. Estimation of Ammonia. The ammonia formed in the hy- drolysis of the protein was estimated by distilling 50 or 100 cc. portions of this solution with a slight excess of magnesium oxide paste (litmus), collecting the distillate in standard acid, and titrat- ing, using methyl red as indicator. The magnesium oxide was first freed from ammonia by boiling with water. Removal of Ammonia. The ammonia was removed from the remainder of the solution by evaporating with a slight excess of barium carbonate. The separate solutions were united and filtered, and the barium-magnesium precipitate carefully washed as before. The excess of barium was removed by dilute sulfuric acid, and the resulting precipitate again carefully washed. The filtrate and wash- ings were evaporated down, made up to one liter, and 5 cc. portions used for Kjeldahl determinations. In this way the amount of nitrogen removed in the second precipitation could be determined. Comparison of the results obtained in the different hydrolysis mixtures shows that the amounts of nitrogen removed by these precipitations were quite constant, and were a little lower for gelatin than for casein. Given in percentage of total nitrogen of the hy- drolysis mixtures, they were: for casein, "first barium sulfate pre- precipitate," 9.76 per cent and 8.86 per cent, "second barium sulfate precipitate," 2.65 per cent and 2.04 per cent; for gelatin, "first barium sulfate precipitate/' 6.87 per cent and 5.79 per cent, "second barium sulfate precipitate," 0.21 per cent and 0.54 per cent. Precipitation of Arginine and Histidine. The cold, slightly acid solution was treated with a hot, saturated solution of silver sulfate, which was added slowly with stirring until the solution con- tained sufficient silver to give a yellow-brown, not a white or pale yellow, precipitate, on removing a drop and testing it with a drop of barium hydroxide solution on a test plate. The brown precipi- tate should form immediately to show excess of silver, as the white silver histidine and arginine precipitate formed will decompose on standing a few minutes and turn brown. In order to be able to follow the nitrogen content of the dif- ferent fractions, silver sulfate rather than silver nitrate must be used here ; but owing to its slight solubility, the operation requires much time, and more water must constantly be added to the mixture, since the silver sulfate keeps separating out as the solution cools. As soon as enough silver sulfate had been added, the cold solution was saturated with finely powdered barium hydroxide in order to pre- cipitate the silver compounds of histidine and arginine. Since the barium hydroxide, even when finely powdered, dissolves slowly, an error may be introduced here, resulting in an incomplete precipita- tion of the arginine, unless care is taken that the solution is actually saturated. This precipitate was filtered off and carefully washed by stirring up several times in a mortar with barium hydroxide solu- tion. Nitrogen in the Lysine and Monoamino Acid Fraction. The filtrate from the above precipitate was acidified with sulfuric acid, freed from silver with hydrogen sulfide, and the filtrates and wash-, ings evaporated down to a definite volume, so that nitrogen deter- minations could be made in suitable aliquot portions. In this way the distribution of nitrogen in the above separation could be fol- lowed. Decomposition of Silver Compounds of Arginine and Histi- dine. The precipitate of the silver salts of histidine and arginine obtained above was suspended in water made acid with a known amount of sulfuric acid, and decomposed with hydrogen sulfide. The precipitate of silver sulphide and barium sulfate formed was 7 carefully washed. It was found that unless special care was taken here, silver was carried over in the filtrate. The thorough decompo- sition of the silver compounds of histidine and arginine was there- fore made certain by saturating the filtrate and washings also with hydrogen sulfide. This precaution was again borne in mind when decomposing these silver compounds separately by hydrogen sulfide in acid solution. The filtrate and washings were evaporated down to remove hydrogen sulfide, made up to 1 liter and Kjeldahl determinations made in suitable aliquot portions. Thus the amount of nitrogen in the arginine and histidine fraction was obtained. Separation of the Histidine Fraction. Several methods have been recommended for separating the histidine from the arginine in the above mixture. (a) In the original Kossel method 1 the separation is based entirely on the difference in the solubility of the silver compounds of histidine and arginine in neutral and in strongly alkaline solu- tions respectively. It is as follows: The solution is freed from sulfuric acid by neutralizing to litmus with barium hydroxide and adding barium nitrate as long as a precipitate is formed. The barium sulfate is filtered off and washed. The solution is concentrated to 300 cc., acidified with nitric acid, if necessary, and treated with silver nitrate until an excess of silver is present. The solution is then exactly neutralized to litmus with barium hydroxide and 5 cc. of a cold saturated solu- tion of barium hydroxide added. If 10 cc. of the filtered solution, when tested with a drop of barium hydroxide solution, give a pre- cipitate which indicates that the silver salt of histidine is not com- pletely thrown down, 2 cc. of saturated barium hydroxide solution are added to the main bulk, and this test is repeated until a test por- tion remains clear. The precipitate of the silver salt of histidine is then filtered off and washed with barium hydroxide until free from nitric acid and the filtrate and washings treated to obtain arginine. It was found difficult to determine, by the above method, when the silver salt of histidine had been completely thrown down. (b) Kossel and Pringle 2 tried to make this precipitation more definite by adding a suspension of barium carbonate instead of *Z. physiol. Chem. (1900-1901), 31; 165-214. 2 Z. physiol. Chem. (1906), 49; 301-324. excess of barium hydroxide to the neutral solution obtained above, warming the suspension on a water bath, and then raising it to the boiling point. After cooling, the histidine silver compound is fil- tered off and washed. No advantage was found in this method and it seems to afford more chance for decomposition to take place. (c) The modification recommended by Osborne, Leavenworth and Brautlecht 1 was found to be the most definite. In it the greater portion of the histidine is first removed from the above mixture by precipitation with mercuric sulfate. It is then reprecipitated as the silver compound. The filtrate from the mercury precipitate still contains a small amount of histidine because of the solubility of the mercury histidine compound. This is separated as the silver salt, as described above, under '(a)'. The work was carried out as follows: The solution obtained by decomposing the silver compound of histidine and arginine was concentrated to about 250 cc., sulfuric acid was added until the solution contained 5 per cent of this acid, and a slight excess of mercuric sulfate solution then added. The precipitate formed was allowed to stand for twelve to twenty-four hours, was then filtered off, washed with 5 per cent sulfuric acid, suspended in water, and decomposed with hydrogen sulfide. The filtrate and washings from the mercuric sulfide which contained the histidine were neutralized with barium hydroxide solution, and barium nitrate was added until no more barium sulfate was precipi- tated. The barium sulfate was filtered off and carefully washed. The histidine was then thrown down again, this time as the silver compound as described under '(a)' above. The filtrate and washings from the mercury histidine precipi- tate which contained the arginine and a small amount of histidine was freed from mercury with hydrogen sulfide, freed from hydro- gen sulfide by evaporation, neutralized, and treated as described under '( a )' to throw down the silver histidine compound. This precipitation was found to be quite definite, and the amount of barium hydroxide solution needed was small. Decomposition of the Histidine Silver Compound. The com- bined precipitates of the silver compound of histidine were sus- . Jour. Physiol. (1908), 23; 180-200. 9 pended in water made acid with sulfuric acid and decomposed with hydrogen sulfide. The silver sulfide was filtered off and carefully washed. The solutions and washings were concentrated and made up to a definite volume so that nitrogen determinations could be made. Preparation of Histidine Solution for Use. The above solu- tion was made alkaline with barium hydroxide and the barium sul- fate filtered off and washed. Excess of barium was then removed by treatment with carbon dioxide and the solution evaporated to dryness. The residue was extracted with hot water, made up to a definite volume and nitrogen determinations in suitable portions made. The amount of histidine present in this solution was then calculated. It was found to be 1.66 per cent of the total protein for casein, and 0.43 per cent of the total protein for gelatin. Osborne 1 reports 2.50 per cent of the total protein for casein, and Hart 2 gives 0.40 per cent of the total protein for gelatin. Decomposition of the Arginine Silver Compound. The filtrate containing the arginine was saturated with finely powdered barium hydroxide and the precipitate of the arginine silver compound so obtained was filtered off and carefully washed by stirring up in a mortar with barium hydroxide and filtering. This washing was repeated until the precipitate was free from nitric acid. The pre- cipitate was then suspended in water made acid with sulfuric acid and decomposed with hydrogen sulfide. The filtrate and washings from the silver sulfide and barium sulfate were evaporated down to a definite volume so that nitrogen determinations could be made. Preparation of the Arginine Solution for Use. The above so- lution was freed from sulfuric acid with barium hydroxide and treated with carbon dioxide until a precipitate no longer formed. It was then filtered, evaporated down to a definite volume and nitro- gen determinations made. From these the amount of arginine present was calculated. The amount of arginine thus calculated was found to be 7.50 per cent of the total protein for gelatin and 3.40 per cent of the total protein for casein. Kossel's 3 figure for gelatin is 9.3 per cent, and Osborne's 4 for casein is 3.81 per cent of the total protein. iJour. Biol. Chem. (1911), 9; 333-353. 2Hart, Z. physiol. Chem. (1901), 33; 358. 3 Z. physiol. Chem. (1900-1901), 31; 204. *Jour. Biol. Chem. (1911), 9; 333-353. 10 This preparation was used in the experiments with the amylase because the separation of the free base was found to be imprac- ticable and the compounds which could be prepared in crystalline form would introduce substances which were not desired in the enzyme work. EXPERIMENTS WITH THE AMYLASE This part of the work is a continuation of investigations which have been carried out in this laboratory on the influence of amino- acids on the hydrolysis of starches by amylases. 1 Since previous work had shown that the purified preparations of pancreatic amylase were the most sensitive 1 it was decided to study one of these. Materials. The preparation chosen was number T-19-B, which had been purified in this laboratory as previously described. 2 The starch was Merck's "soluble starch according to Lintner," washed nine times with ordinary distilled water and six times with triply distilled water. Moisture and acidity determinations were made, the latter by careful titration with 0.0 IN sodium hydroxide, rosolic acid being used as indicator. Ordinary distilled water redistilled twice, (first from alkaline permanganate solution and then from a very dilute solution of phos- phoric acid through a block tin condenser) was used in making up all solutions and starch pastes, and for the final rinsing of all glassware. Activating solutions were made up with sodium chloride and disodium phosphate which had been recrystallized twice from dis- tilled water and once from triply distilled water. Two preparations of arginine from gelatin and three prepara- tions of histidine (one from casein, one from gelatin, and one pur- chased from the Special Chemicals Company) were employed. The cystine had been prepared by another worker in this laboratory, 3 The other amino-acids were purchased products, glycine (Eimer and Amend), phenylalanine (Kahlbaum), and tryptophane (Special Chemicals Company . Accurately weighed or measured portions of these amino-acids were dissolved in 100 cc. of triply distilled water and titrated with . Am. Chem. Soc. (1919), 41; 1866-1873, and unpublished work. 2 Ibid (1919), 41; 1855-1862. 3 Alice Thompson Merrill, Dissertation (1921). 11 0.01 N sodium hydroxide or 0.01 N hydrochloric acid, rosolic acid being used as indicator. The hydrogen-ion concentrations of the starch pastes containing the neutralized amino-acids were then veri- fied by electrometric and colorimetric determinations. Method. Preliminary experiments showed that the gravimetric method worked out in this laboratory for determining the saccharo- genic power of the enzyme 1 could not be used in the presence of arginine and histidine because of their interference with the deter- mination of reducing sugar by Fehling's solution. This had also been found to be true with cystine. For this reason the amyloclastic power of the amylase was measured instead. The procedure followed was based on the method of Wohlge- muth, 2 and has been previously used and described in this labora- tory. 3 Enough starch to make 600 cc. of a 1 per cent starch paste was weighed out, mixed with 50 cc. of triply distilled water and poured into 100 cc. of boiling water. The paste was boiled for three minutes, cooled, and 25 cc. poured into each of six 100 cc. cylinders. To each of these were added the proper activating agents (5 cc. molar sodium chloride and 2.5 cc. M/50 disodium phosphate), enough 0.0 IN sodium hydroxide solution to neutralize the acidity of the starch, and the amino-acid to be tested, which was also properly neutralized. The mixture in each cylinder was then made up to 100 cc. with triply distilled water and carefully stirred. Forty-two clean ? dry test tubes were placed in a special wire frame basket in a bath of ice water and carefully measured por- tions of enzyme solution introduced into each by means of a 1 cc. pipette which was accurately standardized and graduated to 0.01 cc. Portions of 5 cc. of one of the starch pastes prepared above were then introduced into each of seven test tubes. This was done care- fully by means of a burette with a very long delivery tip reaching to the bottom of the test tube. In this way the lodging of any of the starch paste on the sides of the tube was avoided. Since the tubes were kept in ice water no measurable reaction took place. The basket of tubes thus prepared was shaken and placed in a Freas thermostat in which the temperature varied only about 0.01 C. After 30 minutes the basket was taken out and placed in the ice water to stop the action. The tubes were thus kept at . Am. Chem. Soc. (1915), 37; 628. 2 Biochem. Z. (1908), 9; 1. "Jour. Am. Chem. Soc. (1915), 37; 634. 12 an average temperature of 40C. for 30 minutes. After a few minutes 0.1 cc. of 0.1N iodine in potassium iodide solution was added to each tube. This was done very carefully by means of a dropping bottle which delivers drops of 0.1 cc. volume. About 20 cc. of distilled water was then poured into each tube and the contents thoroughly mixed. Each set of tubes containing the same amount of the same starch paste with varying amounts of enzyme was then observed for the end point; that is, the tube of lowest enzyme concentration which is definitely red and shows no blue or violet color due to starch.* To obtain the value of the amyloclastic power of the enzyme, the weight of the 1 per cent starch paste, 5000 mg., is divided by the weight in milligrams of enzyme present in the tube showing the Wohlgemuth end point. Blank determinations were always made in each set ; that is, starch pastes neutralized and containing the proper activators but no amino-acids were used as the standard each time, since results vary slightly from day to day due to experimental error and deterioration of the enzyme in solu- tion. Data of Typical Experiments. Table I shows the influence of 50 milligrams of arginine and 50 milligrams of histidine on the amyloclastic action of the amylase. It will be seen that the arginine has a distinct "activating" influence, while the histidine has not. These results were confirmed by other experiments. *The terms used in describing the colors are those of the Milton Bradley Standard Color Chart as given by Mullikin in his "Identification of Pure Organic Compounds" 13 Sw 1 a . a 1 a ! c* o^ Ptj FH ' o o o o a i 8)73 * I M o 1 1 09 a I t- o o a; a> * * &? S ^ 0) O "3 V ^ P3 ^ ^ .s I o 8 "8 $8 -P73 v a> i -2 OJ ^ N o [o V ^o V [o V 73 > O | H > > > > 03 -M 4^ ^J 49 j^> ,3 O o o 5 in> o [o |o [o "o [o 1 o I l ? I s 1 i ? 4 1 , [o "o [o [o P > p > > > > s M -p ^ o d) i 1 0> p 0> M ^ s ^ o o 1 o O o PH ua" ^r ^r iO to* CO iH rH (JsJ o^ CO t- t> CO CO "w o Arginine 50 mg. (Gelatin-a) : ||! Histidine 50 mg. (Casein) Histidine 50 mg. (Purchased) 'S ^ S H -.. <-> ~ -~ ^ ^ G u,_, bo ~ -*-> bjQ O -J U^ 5 | a rt oJ o *- y < ) r-; 5 03 ,Q X +J C -> > rt S S '-S * '43 M ^ rt S 4* O cfl cti o J3 -fi j - - 1 -S x 14 < C O g o 1 a) i r PQ 8 g .s-s - * o ^ C fl r rt o> "p -5 _c too 2 ^ > rt w re C 2 CO i stsl-s VH TO ^2 rt rj ^ B.Q. O* O -B^ T3 I 17 bjO I 0) .^H ' M 8*8 ' 8-8 ' H o S I 1 r PQ PQ PH S - s 'S OT -^o^ -^ M LO W M IO "IS ti rch Histidine 50 (Gelatin) a a 5 -Cl Q in s g a > -3 o> i< ^ X - 'So T3