EXCHANGE A Study of the Initial Velocity in the Hydrolysis of Sucrose by Invertase. DISSERTATION Submitted in Partial Fulfillment of the requirements for the Degree of Doctor of Philosophy in the Faculty of Pure Science of Columbia University. BY HAROLD LESTER SIMONS, A.B., A.M. NEW YORK CITY 1921 A Study of the Initial Velocity in the Hydrolysis of Sucrose by Invertase. DISSERTATION Submitted in Partial Fulfillment of the requirements for the Degree of Doctor of Philosophy in the Faculty of Pure Science of Columbia University. BY HAROLD LESTER SIMONS, A.B., A.M. NEW YORK CITY 1921 TO MY FATHER AND MOTHER ^51732 ACKNOWLEDGMENT The author desires to express his gratitude to Professor J. M. Nelson, at whose suggestion this work was carried out. To his fruitful suggestions and to his kindly encouragement, this research owes what- ever merit it may possess. ABSTRACT OF DISSERTATION 1. What was attempted ? 2. In how far were the attempts successful? 3. What contribution actually new to the science of Chem- istry has been made ? 1. In view of the fact that many investigators have ex- trapolated the initial portion of the curve representing the hydrolysis of sucrose by invertase by assuming that the func- tion was initially linear and due to the importance of knowing the actual course of the inversion at its start, an attempt has been made to determine : (a) Whether this condition actually exists ; (b) In the case of its actual existence, whether it is due to lack of sufficiently great experimental precision or is due to some peculiarity in the reaction. 2. It has been found that (a) In general, the initial course of the curve represent- ing the hydrolysis is linear ; (b) Under certain conditions, however, the velocity ac- tually rises to a maximum ; (c) The observed linear part of the curve is due mainly to a peculiarity of the reaction. 3. It has been shown that (a) By actual measurement the velocity of the inversion of sucrose by invertase is, in general, constant at the start. Thsi is the usual case even in the presence of added substances. However, under certain conditions, the velocity has been ob- served to go through a maximum before the gradual decrease sets in ; (b) This constant or increasing velocity is only evident at the beginning of the hydrolysis ; 7 (c) These observations particularly in view of the fact that the initial velocity at times increases strongly indicate that invertase action involves a series of consecutive reactions ; (d) Since the velocity often increases to a maximum dur- ing the earlier part of the reaction, the initial velocity cannot be taken as a true measure of the activity of the invertase. In the light of this fact, the theory of invertase action proposed by Colin and Chaudun (which depends upon the assumption that the rate of combination between sucrose and invertase is infinite) is untenable. This criticism likewise applies to the theory advanced by Michaelis and Menten which fails to ac- count for some other facts disclosed by this investigation. A STUDY OF THE INITIAL VELOCITY IN THE HYDROLYSIS OF SUCROSE BY INVERTASE In determining the activity of hydrolytic enzymes, like invertase, the question arises as to what extent the velocity of the hydrolysis of the substrate can be taken as a measure of the activity or, in other words, how much of the enzyme pres- ent in the solution is in the active condition. Henri 1 proposed Kt= (1 +na)ln- -+ (m n)x (1) a x as an equation for representing the kinetics for the hydrolysis of cane sugar by invertase. In his derivation of this equation (1), he like many others (Brown 2 ) assumed one unit of inver- tase to combine (reversibly according to the Mass Law) with one mole of cane sugar and also with the inverted cane sugar as it was formed in the reaction. The terms m and n in the equation are the equilibrium constants of the cane sugar in- vertase and inverted cane sugar invertase compounds re- spectively. The K is the velocity coefficient of the hydrolysis, and a x and x represent the concentrations of the cane sugar and inverted cane sugar at the time t. He also assumed that the velocity of combination between the enzyme and sugars is infinite when compared to the velocity of hydrolysis. Henri was unable to obtain experimental values for the mass law constants (m and n) and therefore resorted to a method based upon trial, assigning to m and n arbitrary values which gave fairly constant values for the velocity coefficient, K. Hudson 3 objected to Henri's results on the ground that the latter had followed the course of the hydrolysis by permit- ting the reaction to take place in a polariscope tube and notic- ing the change in rotation of the sugar solution from time to time. The rotation values obtained in this way involve an error, since the GJ " A -Ex per in A - d- ent L Z I JT ^/ Y 30 60 90 120 150 ISC Minutes Fro! 19 place as rapidly as possible) cane sugar per 100 cc. of the solu- tion and allowed the hydrolysis to continue. The velocity hy- drolysis of the added portion of cane sugar was then compared with the velocity of a 10% cane sugar solution (A) after the latter had passed the 50% hydrolyzed stage. The change in rotation of the solutions and the corresponding times are given in Table IV, and the corresponding graphs in Figure III. The calculated rotation of the 10% cane sugar solution A when 50% hydrolyzed was 3.80. The rotations given in Table IV were determined by adding 5 cc. of a sodium car- bonate solution to 20 cc. of the sugar solution and then ex- amining it in the polariscope with a 200 mm. tube. The inver- tase correction for the rotations in this set of experiments was negligible. By subtracting 3.80 from the initial rotation of solution A, 10.64, 6.84 is obtained as the change in rotation corresponding to the 50% hydrolysis stage. Locating this point, 6.84, on the curve in Figure III and reading the corre- sponding time co-ordinate, the latter was found to be 82.0 min- utes. The 5% hydrolyzed cane sugar solutions to which 5% more cane sugar had been added (that is solutions B I and B IL in Table IV) had, in the case of B I a rotation 3.44 after the reaction had been in progress for 4.0 minutes. Solution A had the rotation 3.44 after it had been undergoing hydrolysis for 87.5 minutes or in other words it took solution A 87.5 82.0 or 5.5 minutes to undergo the same extent of hydrolysis as solution B I had undergone in 4.0 minutes. Similarly it was found that solution B II required only 9.5 minutes to underg'o the same degree of hydrolysis as solution A underwent in 11.3 minutes. This again shows, as was demonstrated by the data given in Table II that the initial velocity, in the case of solu- tion B, is greater than the velocity in such a solution, as A, where the reaction has been in progress for some time. But the data in Table IV also shows, as can be seen in Figure III, that the hydrolysis curve of solution B is super-imposable upon that of solution A and that the time-element is only apparent dur- ing the initial stage of a hydrolysis of cane sugar by invertase. Besides the experiments of Table IV, another set was exam- ined in which 2.5 grams of cane sugar per 100 cc. solution in- 20 stead of 10 grams were used and the velocity of hydrolysis be- yond the 50% stage was compared with that of a solution con- taining 1.25 grams of cane sugar and 1.25 grams of inverted sugar per 100 cc. solution. These curves were found to coin- cide also. TABLE IV. Part A. 10 grm's. cane sugar per 100 cc. Temp. 25. Hydrogen ion cone. 10 i - 2G (200 mm. tube). I. II. Time in minutes Rotation 0.0 10.62 2.5 10.44 14.0 9.35 46.0 6.60 91.5 3.27 126.5 1.26 175.5 0.66 00 3.06 Time Rotation 0.0 10.64 17.0 9.10 59.0 5.45 101.5 2.58 144.5 0.46 176.0 0.70 222.0 1.81 260.0 2.20 CO 3.04 Part B. 5 grms. cane sugar and 5 grms. inverted cane sugar per 100 cc. Temp., Invertase cone, and hydrogen ion cone, same as in A. II. Time Rotatioi 0.0 ( 83.5) 4.0 ( 87.5) 3.44 20.5 (104.0) 2.46 55.0 (138.5) 0.75 100.0 (183.5) 0.89 146.0 (229.5) 1.90 219.0 (302.5) 2.60 co 3.09 Time Rotation 0.0 ( 83.5) 9.5 ( 93.0) 3.08 42.5 (126.0) 1.25 71.0 (154.5) 0.00 102.5 (186.0) 1.00 132.5 (216.0) -1.68 164.0 (247.5) 2.17 225.5 (309.0) 2.65 Does Invertase Action Involve Two or More Consecutive Reactions? It will be observed upon inspection of the curves, in Figure I, part C, for the 1% cane sugar solutions containing 3% and 21 4% added glucose that they are slightly convex to the time axis at the start, or in other words, the initial velocity of hy- drolysis under these conditions seems to increase. This in- creasing effect is so small, however, and the measurements involve the determination of such small absolute changes in the extent of the hydrolysis that it might be attributed to pos- sible experimental errors. It was decided therefore to repeat these determinations in somewhat different manner. Instead of using* the polariscope which is accurate in this case to 1 milligram of hydrolyzed cane sugar in 50 cc., a copper reduc- tion method (for details see the experimental part of the paper) which has a higher degree of precision, 0.1 milligram of hy- drolyzed cane sugar per 50 cc. of solution, was adopted. In order to avoid the large amount of cuprous oxide that would be formed if the copper method were used in the case of the above 1% cane sugar solution containing 3% or 4% of added glucose, a 0.5% cane sugar solution, containing no add- ed glucose, was used. This solution had about the same speed of hydrolysis as the 1% solution under the above conditions. A hydrolysis of a 1% cane sugar solution without any added glucose was followed also by means of the copper method for the sake of comparison, Part C, Table V. In Table V are given the results obtained and these results are plotted also in Figure IV. Dr. Vosburgh, of this laboratory, has shown (his results are to be published shortly) that certain invertase preparations when acidified with hydrochloric acid to adjust the hydrogen ion concentration of the solution, do not always give repro- ducible results. For this reason, the hydrogen ion concentra- tion of the 0.5% cane sugar solution was adjusted in two ways. The results given in Part A of Table V were obtained by using hydrochloric acid, and those in Part B by using an sodium ace- tate-acetic acid buffer mixture. In this way, if the velocities obtained in the two cases were the same then any abnormality in the velocity of hydrolysis that might appear cannot be at- tributed to the method of adjusting the hydrogen ion concen- tration of the solutions. 22 TABLE V. Part A. 0.5% cane sugar solution. 2 cc Invertase per 100 cc. solution. Temperature 25. Hydrogen ion, 1CM- 4 moles per liter and adjusted by means of HC1. Mg. cane sugar Time Mg. Cu. hydrolyzed per 50 cc. 1.03 11.28 4.02 2.14 22.95 9.30 3.01 30.34 13.44 4.03 40.37 18.14 5.03 49.10 22.22 6.03 . 57.52 26.20 7.00 65.46 30.09 8.00 74.15 34.26 9.04 82.25 38.23 10.01 90.18 42.19 11.02 98.16 46.20 Part B. Same solution and conditions except sodium acetate acetic acid used for adjusting the hydrogen ion concentration; Mg. cane sugar Time Mg. Cu. hydrolyzed per 50 cc. 1.03 12.62 4.30 2.02 22.27 8.90 3.04 31.56 13.43 4.03 40.56 17.90 5.03 48.95 22.02 6.03 56.59 25.60 7.00 64.25 29.35 Part C. l c / c cane sugar solution, other conditions same as for B. Mg. cane sugar Time Mg. Cu. hydrolyzed per 50 cc. 0.52 13.78 3.22 1.06 20.20 6.59 1.56 27.17 9.82 2.08 33.87 13.20 2.65 41.19 16.75 3.23 49.37 20.67 3.88 58.86 25.72 4.41 65.57 28.86 From the data in Table V and the shape of the curves in Figure IV, it is evident again that this increase in the initial velocity 23 of hydrolysis, at least in the case of the 0.5% cane sugar solu tion, shows up just as in the case of the curves Part C, Figure I 48 42. 18 A Part A 6 C 6 9 Minutes FIG.N 12 15 To gather more evidence still on this point, the results ob- tained by other investigators from dilute sugar solutions were examined and plotted in the same way. Here again this same increase of the initial velocity was found to occur. Thus ex- periments 31 A and 31 B of Nelson and Vosburgh 6 in which a 0.4% cane sugar solution was used and similar experiments 7, p. 337, and F, p. 341- described by Michaelis and Menten (loc. cit.) in which 0.178% and 0.821% cane sugar solutions 24 respectively were used, all showed this increase in the initial velocity. The Retarding Influence of Very Small Amounts of Glucose on the Velocity of Hydrolysis. TABLE VI. Cone, cane sugar = 1 g. per 100 cc. Cone. Invertase=:0.5 cc. per 100 cc. Cone. Hydrogen ion = 10- 4 - 4 . Temperature = 25. Cone, of Rotation after Change Glucose % Init. Rotation 50 min. X100 0.0 358.830 358.25 58 0.5 359.95 359.43 52 1.0 1.08 0.60 48 2.0 3.34 2.92 42 3.0 5.60 5.23 37 4.0 7.87 7.52 35 \ \ ^^ \ ^^ 63 \ \ \ t \ \ \ 1" \ x \\ \\ \\ Vb v\ 1" \\ \\ ^ \\ r \ i i" \ N ^^ 33 123 Cone. Glucose in Grams per 100 cc. FIG.Y 25 In Table VI is recorded the data obtained in studying the retarding influence of varying amounts of added glucose upon the rate of hydrolysis of a 1% cane sugar solution. It will be observed, especially by the inspection of curves in Figure V, a graph of the data, that, although the retardation is increased by augmenting the glucose concentration, yet each successive increment of the retardant has less and less effect. Thus, the measured effect of 0.5 grams of glucose at the beginning of the reaction is 58 52 or 6 units (see last column in Table VI) while when 3 grams were added then a decrease of 21 units was observed. In the first instance, the specific retardation (the specific retardation is defined as the influence of each unit of 1 gram of glucose under the conditions of the experiment) was 58 52 or 12 and in the last 58 34 or 7. Furthermore 0.5 ~T~ it must be borne in mind that at the time, 50 minutes after the start of the reaction*, when the readings were made, there was present in the solution containing no glucose about 0.17 grams per 100 cc. of invert sugar formed in the hydrolysis that had taken place up to this time. If the retarding influence of the invert sugar is considered as practically the same as that of glucose, then the~ specific effect of the 0.17 grams of invert sugar should be greater than that of 0.5 grams of glucose, since the retarding effect increases with decreasing amounts of the monoses present. If this is true, the value obtained for the "initial velocity of hydrolysis" must be too low due to the greater specific retardation of small amounts. This merely points out that the measured retardation effects of the small amounts must be too low and that in the light of this the true curve representing the retardation should rise very rapidly as it approaches the velocity axis and may possibly be asymp- totic to it. As a consequence, the probable character of the true retardation curve is indicated by the dotted line in Fig- ure V. * It will be readily seen that, if there be no time effect at the start of ^ the reaction, the change in rotation after 50 minutes will be on the initial linear portion of the hydrolysis curve with this concentration of enzyme. This would be a consequence of the results of Nelson and Vosburgh 6 , who have shown that the velocity of the reaction is propor- tional to the invertase concentration. 26 The retarding effect of the invert sugar, as it is formed, there- fore should cause a hydrolysis curve of a cane sugar solution to fall off rapidly from the start. This is, however, not the case, as has been shown above. A tentative explanation there- fore suggests itself, that some other phenomenon besides re- tardation occurs which tends to offset the latter, thus giving rise to the constant or slightly increasing initial velocities ob- tained in this work. EXPERIMENTAL Preparation of Invertase. The invertase was prepared from yeast which had been allowed to autolyze. The filtered solu- tion had been kept in a stoppered bottle for about three years previous to its purification. In general, the procedure followed was that described by Nelson and Born 7 . It was found that treatment of the autolyzed and filtered yeast solution with am- monia (stirring to prevent local alkalinity) until it was just short of being neutral to litmus, was very helpful in removing the precipitate obtained by the lead acetate. The invertase was not precipitated again after dialysis but was treated with toluene, bottled and kept in an ice-box. No loss in activity was noticed throughout the period of the investigation. Cane Sugar. The cane sugar used was a good commercial grade such as Domino or Jack Frost which had been recrystal- lized from alcohol according to the method of Cohen and Com- nielin 8 and dried in a 0.04 mm. vacuum at 50 over sulphuric acid. Glucose and Fructose. The glucose was a very pure com- mercial grade, obtained from the Corn Products Co., N. Y., and was recrystallized from acetic acid according to the method of Hudson and Dale 9 . It was washed with alcohol and dried at 50 over sulphuric acid at 0.04 mm. In order to remove the last traces of acetic acid, the drying was repeated over stick caustic soda. The fructose was twice recrystallized from acetic acid ac- cording to Vosburgh 10 . It was noticed, that, if the alcohol washings were combined with the filtrate and if the mixture 27 were placed in the ice-box, a large quantity of fructose could be recovered after about a week's standing. The fructose was dried in the same manner as the, glucose, except that the tem- perature was maintained at 30. To prevent decomposition and absorption of moisture, the sugars were kept in the dark in stoppered bottles inside a dessicator. They all exhibited the correct rotations. The invert sugar used in this work was made up from equal quantities of glucose and fructose. Preparation of Solutions, Table I. The method, with a few modifications, described by Nelson and Vosburgh 6 was used for the experiments given in Table I. 200 cc. of a sugar solu- tion, of twice the concentration, required, were added to an equal volume of invertase solution, also of twice the required concentration. A stream of air was blown through the solu- tion to insure rapid and thorough mixing. Both the cane sugar and invertase solutions had been kept in the thermostat for at least 2 hours before mixing to permit them to acquire the cor- rect temperature. Solutions, Table II, III and V. The cane sugar solutions in this case, were made up by the aid of specific gravity tables 11 and corrected for 25 according to Schonrock's formula 11 . The required amount of powdered cane sugar was placed in a non- sol bottle. Water and the required amount of buffer were added so that on the addition of a definite volume of stock in- vertase solution (18.05 cc. or some other selected quantity) the final volume would be exactly 900 cc. Vigorous agitation by a current of air was resorted to. Solutions, Table VII. The same procedure was followed as described for solutions, Table I, save that 25 cc. portions of sugar and invertase solutions were used instead of 200 cc. Hydrogen ion adjustment. All the experiments were car- ried out at a hydrogen ion concentration of 1CH- 4 moles per liter. For experiments described in Tables I and V, Part A, 0.01 molar hydrochloric acid was used according to the method of Nelson and Vosburgh 6 (that is comparing the solutions 28 colorimetrically with citrate solutions that had been standard- ized by the electrometric method). In the electrometric stand- ardization of the citrate solutions, a saturated potassium chlor- ide-calomel half cell and a saturated potassium chloride bridge were used (Fales and Mudge 12 ). In all the other solutions the hydrogen ion concentration was adjusted by means of sodium acetate-acetic acid buffer according to Michaelis 13 . This latter method was checked and found satisfactory. Measurements of Volume and Time. The measuring flasks, burettes and pipettes were carefully calibrated. In the experi- ments described in Tables II, III, and V, the pipettes used had a time delivery from 8 to 12 seconds in order to eliminate any error in ascertaining the time of stopping the reaction when taken as a mean. The 18 cc. invertase pipette delivered in 7.5 seconds. They were all held vertically and removed when the free flow ceased. It was found that an operator can duplicate results by this method with a precision of better than 0.01 cc. In the other experiments ordinary calibrated pipettes were employed. The time of delivery of the pipettes and the length of time of the reactions (that is the time from the introduction of the enzyme to the interruption of the hydrolysis) were follow- ed with a stop watch which could be read< to 0.2 of a second and therefore the time intervals in all the Tables, except I, are precise to at least 0.01 minute. All weights are so-called "weights in air." Stopping the reaction. The reaction was stopped by run- ning the 50 cc. samples from the reaction bottle in the thermo- stat, into 5 cc. of 0.2 molar sodium carbonate. The procedure was reversed in the experiments of Table VII. In those of Table I this operation was done in 60 cc. flasks and then made up to volume. Determination of Extent of Hydrolysis. In all the experi- ments save those recorded in Table V, the degree of hydrolysis was determined in a 400 mm. jacketed tube which had been calibrated. The temperature was maintained at 25.00 0.05 29 by pumping water through the tube from the thermostat. The hydrolyses were all conducted at 25.043 0.005. A Schmidt and Haensch triple field instrument at a half shadow angle of 2 was used in conjunction with a 1,500 candle power quartz mercury .vapor lamp. The light was filtered through an Eastman No. 74 Wratten filter which had been test- ed with a Lummer-Brodhun spectrophotometer. This test showed that practically none of the mercury lines was trans- mitted except 546 ^ ^ Readings could be made after some practice with a precision better than 0.01. Determination of Initial Angle. In obtaining the initial angles, Table I, 25 cc. of water and 25 cc. of cane sugar solu- tion of double strength were run into 5 cc. of sodium carbonate solution, and this made up to 60 cc. and read in the polariscope. A correction was then made for the rotation that the invertase would have if present. In the other experiments, instead of the 25 cc. of water, 25 cc. of a double strength invertase solu- tion were employed so as to conform to the rest of the readings in the same experiments. Copper Method. The method of Thomas and Quisumbing of this laboratory, which is to be published soon, was used for determining the degree of hydrolysis in Table V. Beakers, containing 50 cc. samples, 5 cc. 0.2 M sodium car- bonate and 25 cc. each of the modified Fehliug's solutions A and B, were immersed in a water bath kept at 80 1. for 30 minutes. The cuprous oxide was filtered off, washed and dissolved in dilute nitric acid. A small amount of sulphuric acid was added to this solution which was evaporated to dry- ness on a hot plate. The copper was determined finally by the thiosulphate method. To construct the calibration curves for "reducing sugar", the following procedure was employed. Standard cane sugar and invert sugar solutions were made up. Definite amounts of these were added to 5 cc. of sodium carbonate into which the correct number of cc. of invertase solution had been run prev- iously. Water was then added so that the final volume would be 55 cc. The analysis was then performed as described above. The addition of the invertase was necessary as it formed a cop- 30 per compound and thus tended to make the results too high unless allowed for. As the amount of this copper compound varied with the concentrations of the other substances present, it could not be corrected for and was therefore included in the calibration mixture. These calibration curves were construct- ed separately for each hydrolysis in order to eliminate any error due to varying composition of the Fehling's solutions. The data for these curves are given in Table VIII. TABLE VIII. For PartA, Table V For Part B, Table V For Part C, Table V Mg. Cane Mg. Cane Mg. Cane Sugar hydrol . Mg. of Sugar hydrol . Mg. of Sugar hydrol . Mg. of per 50 cc. Copper per 50 cc. Copper per 50 cc. Copper 0.00 2.61 0.00 2.73 0.00 6.02 2.00 5.29 2.00 6.51 2.00 11.09 4.00 11.80 4.00 11.93 4.00 15.47 6.00 16.45 6.00 16.34 6.00 19.08 8.00 20.73 8.00 20.49 8.40 23.77 10.00 23.98 10.00 24.63 9.60 26.75 20.00 44.33 15.00 35.68 15.00 37.46 30.00 65.25 20.00 44.87 20.00 48.11 40.00 85.85 25.00 55.40 25.00 57.40 50.00 105.62 30.00 65.67 30.00 68.01 35.00 75.05 The points on the curves were so chosen that they corre- sponded to every two milligrams of cane sugar hydrolyzed up to the first 10 milligrams and to every five or ten milligrams of hydrolyzed cane sugar thereafter. SUMMARY (1) The initial velocity of the hydrolysis of cane sugar in the presence of invertase was found generally to be constant for a considerable period after the beginning of the reaction. (2) A sucrose solution, containing added invert sugar, hy- drolyses with a different initial velocity than that manifested during the reaction by a partially hydrolyzed cane sugar solu- tion of the same composition. (3) This difference in influence upon the velocity of the hy- drolysis, between invert sugar added to the reaction at its be- 31 ginning 1 and that formed during the hydrolysis, renders the method of Michaelis and Menten for determining the dissocia- tion constants of the so-called sugar-invertase compounds valueless. (4) It was found that the initial velocity of hydrolysis of dilute sucrose solutions or solutions to which a considerable amount of glucose had been added appeared to increase for a short period after the beginning of the reaction. (5) The specific retardation due to glucose decreases as the concentration of hexose is increased and finally reaches a minimum. This indicates that the true initial velocity may be very great and (when the facts in section 4 are considered) that the hydrolysis probably consists of a series of consecutive reactions. 32 BIBLIOGRAPHY 1 Henri Lois generates de 1'action des diastases. Paris (1903). - Brown/. Chem. Soc., 81, 373, (1902). 3 Hudson/. Am. Chcm. Soc., 30, 1160, 1564, (1908). 4 Armstrong Proc. Roy. Soc., 73, 516, (1904). 5 Michaelis and Menten Biochem. Zeit., 49, 333, (1913). " Nelson and Vosburgh /. Am. Chem. Soc., 39, 797, (1917). 7 Nelson and Born/. Am. Chem. Soc., 36, 393, (1914). 8 Cohen and Commelin Z. physik. Chcm., 64, 29, (1908). 9 Hudson and Dale/. Am. Chem. Soc., 39, 790, (1917). 10 Vosburg-h /. Am. Chem. Soc., 42, 1696, (1920). 11 Browne Handbook of Sugar Analysis, (1st edition), page 30 and appendix, page 1. 12 Kales and Mudge /. Am. Chem. Soc., 42, 2434, (1920). 12 Michaelis Die Wasserstoffionenkonsentration, page 184. 13 Colin and Chaudun Compt. Rend., 167, 338, (1918). VITA Harold Lester Simons was born in New York City on July 15, 1897. He graduated from the College of the City of New York in June, 1917, receiving the degree of Bachelor of Arts. He entered the Graduate School of Pure Science of Columbia University in July, 1917, and received therefrom the degree of Master of Arts in June, 1918. He was Laboratory Assistant in Chemistry at that institution from February, 1918 to February, 1919. He was co-author (with Dr. H. L. Fisher) of a paper on "Dimethyl Tartronate" published in the Journal of the American Chemical Society, 43, 628, (1921). THXS DATE AN INITIAL PINE OF 25 CENTS APR 9 1938 '46 DL LD 21-95w-7,'37 Gay lord Bros. Makers Syracuse, N. Y PAT., " "M YC 39876 151782 55" UNIVERSITY OF CALIFORNIA LIBRARY 1 /