Influence of Certain Amino Acids upon the Enzymic Hydrolysis of Starch DISSERTATION Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Faculty of Pure Science, Columbia University. By FLORENCE WALKER, A.B., A.M. NEW YORK 1922 Influence of Certain Amino Acids upon the Enzymic Hydrolysis of Starch DISSERTATION Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Faculty of Pure Science, Columbia University. By FLORENCE WALKER, A.B., A.M. N NEW YORK 1922 ACKNOWLEDGMENT This investigation is a continuation of the study of amylases begun by Professor H. C. Sherman in 1907. The author is greatly indebted to Pro- fessor Sherman for many helpful suggestions re- ceived from him during the course of this work. INFLUENCE OF CERTAIN AMINO ACIDS UPON THE ENZYMIC HYDROLYSIS OF STARCH In 1893 Effront 1 stated that asparagine accelerates the hydrolysis of starch by malt and taka-diastase. A few years later 2 he obtained similar activation by addition of certain proteins and of a boiled cold water extract of barley. In 1904 he reported 3 that asparagine aspartic acid, hippuric acid, creatin, creatinin and the peptones in- creased the action of malt extract, while succinamide, the amines and their salts, and acid amides generally act unfavorably. This he found for several starches of different origin. He also found, how- ever, that the more favorable the conditions for the production of an optimum amount of sugar, the less marked is the effect of the amino acids. * Ford*, working with malt, found asparagine to be without effect on the activity of the enzyme. The apparent activation by amino acids and acid salts obtained by other investigators, he ascribes to the neutralization by these compounds of alkaline impurities in the starch. According to Terroine and Weill 5 saccharification by pancreatic juice is greatly accelerated by alanine, glycine, leucine, valine, histi- dine, arginine, tyrosine, phenylalanine, aspartic acid and glutamic acid. The activating power which they found the digestion products of protein to possess is, they think, most probably due to the amino acids formed. More recently Rockwood 6 has investigated the effects of nitro- geneous substances on the hydrolysis of corn starch by saliva and concludes that glycine, tyrosine, anthranilic acid and its meta and para isomers, aspartic acid, hippuric acid, proteins (serum albumin and gelatin) and amines of the methane series increase saccharifica- tion, whereas the amides (acetamide, propionamide and urea), sul- phanilic acid, asparagine, succinamide and succinimide show no such *Mon. sci., 41, 266 (1893). *Compt. rendu., 120, 1281 (1895). 'Bull. soc. chim., [3] 31, 1230 (1904); Mon. sci., [4] 18, 561 (1904); Compt. rendu. soc. biol., 57, 234 (1904). 4 J. Soc. Chem. Ind., 23, 414 (1904). 5 Compt. rendu. soc. biol., 72, 542 (1912). "Jour. Amer. Chem. Soc. 39, 2745 (1917). . .. 478719 4 Influence of Certain Amino Acids effect. He also found that glycine and aspartic acid activate pan- creatic extract. Because of the lack of agreement among previous workers as to whether amino acids do or do not activate amylases, and in view of the fact that past investigations have seldom covered more than one enzyme, a systematic study of the influence of amino acids upon the action of several amylases, in purified as well as in their natural or commercial forms, seemed desirable. An attempt has been made in the present work to throw some light on the manner in which the amino acids act. EXPERIMENTAL Apparatus and Materials. The glassware was of the best quality, carefully selected and when not in use was kept filled with water. Just before using, it was washed with soap, rinsed ten times with tap water, three times with ordinary and once with triple distilled water. A Freas thermostat bath with a variation of + 0.005 was used for all digestions at 40 C. Digestions at other temperatures were carried out in a small bath kept constant within +0.15 by means of an Ostwald regulator. Merck's "soluble" starch according to Lintner purified by washing 9 times with ordinary distilled and 6 times with triple distilled water has in all cases been used as substrate. The salts employed as activators were C. P. recrystallyzed several times. The amylase preparations and other enzyme containing materials tested were (1) pancreatic amylase preparations Nos. 58, 59, 60, 77B, 81B and 11B, (2) commercial pancreatin No. 8, (3) malt amylase preparation No. 155, (4) malt extract, (5) aspergillus amylase preparations Nos. 22, 22b, and 23, (6) commercial takadiastase, and (7) fresh saliva. Triple distilled water was used for making starch disper- sions of activators, enzymes, etc., and for rinsing all glassware. Method. The method 7 of testing the influence of the amino acids is briefly as follows. An amount of air-dry starch equivalent to the required amount of anhydrous material is weighed out, mixed with a little cold water, dispersed by pouring into boiling water (about 80 c.c. per gm. of starch) and boiled for about 3 minutes. This is i Sherman, Kendall and Clark, Jour. Amer. Chem. Soc., 32, 1082 (1910). upon the Enzymic Hydrolysis of Starch 5 transferred to 100 cc. cylinders, neutralized with 0.01 N sodium hydroxide solution and the salts 8 most favorable for the action of the amylase added. The dispersions are then made up to 100 c.c. so that the concentration of starch is exactly 1 per cent, mixed thoroughly by stirring and placed in the 40 bath to reach the desired temperature. In the meantime, the enzyme solution is prepared and the required amount pipetted into dry flasks. The starch dispersions are then poured into the flasks containing the enzyme at intervals of 15 seconds and the flasks placed in the 40 bath. At the end of 30 minutes, enzymic action is stopped by pouring 50 c.c. of Fehling solution into the digestion mixtures, at intervals of 15 seconds and in the same order in which the starch was poured on the enzyme. The amount of reducing sugar formed is determined by immersing the flasks in a boiling water bath for 15 minutes. The cuprous oxide is filtered into weighed Gooch crucibles, washed with hot water, alcohol and ether, dried at 100, and weighed. Glycine and alanine being quite soluble were dissolved in a small volume of water and added to the starch paste, after it was poured into the cylinders and before being made up to volume. Since tyrosine and phenylalanine are difficultly soluble, the amount of each used was added to the water in which the starch was dispersed and boiled with it. To show whether this variation in procedure affected the action of the amino acid on the enzyme, digestions were carried out in which equal amounts of asparagine were added before boiling in some cases and after cooling in others. Activation due to asparagine was the same in both cases. The same test was made with aspartic acid with the same result. In these experiments the amino acids were made neutral to rosolic acid with 0.01 N sodium hydroxide solution. MEASUREMENT OF THE INFLUENCE OF DIFFERENT AMINO ACIDS Tables I-IV show the influence of carefully neutralized glycine, alanine, tyrosine and phenylalanine, added separately and in com- bination with a second amino acid, upon the rate of hydrolysis of "soluble starch" by different enzymes. The reducing sugar formed 8 Sherman, Thomas and Baldwin, ibid., 41, 231 (1919). Pending further investigation the substrate is prepared in the same manner for the action of saliva as for pancneatic amylase. 6 Influence of Certain Amino Acids by enzymic hydrolysis is chiefly maltose, but since small amounts of glucose may also be present the results are stated in terms of the weight of cuprous oxide resulting from the reduction of Fehling solution by the sugar or sugars present. The amounts of enzyme used in the experiments were so regulated as to result in the trans- formation of about Ys of the starch into sugar. TABLE I Effect of Glycine and Glycine Plus Aspartic Acid on the Enzymic Hydrolysis of Lintner Soluble Starch Amino Acid, Mg. Cuprous Oxide, Mg. d 1 tj rt o * 0^ !a.s M^- .2 rt .1 3 III P >-> rt 1 J "K N 11 11 ll^ rt "xJ ~ o TL fi > ** **> M *> >< 5 rt j? &y l ss E = *(? 13 |i l"rt:z O Oft VI 7M 31 ! UH^ 246 227 316 260 277 222 292 50 280 243 334 270 286 226 292 100 284 245 341 273 283 224 292 50 279 242 344 269 280 223 289 50 50 279 247 334 268 281 224 292 Activation due to glycine 38 18 25 13 9 4 TABLE II Effect of Alanine and Alanine Plus Glycine on the Ensymic Hydrolysis of Lintner Soluble Starch Amino Acid, Mg. Cuprous Oxide, Mg. u B V ^ TO (fl &Z3 II a ^-> oj w &J2 ;- 1 'o jh ill II _> It Sa "rt IE o rt^ < 5 PnPH^J Cfl PH< a |J 7! s-5. 282 294 323 248 252 261 287 50 318 317 353 262 266 273 299 50 316 312 352 256 255 263 291 25 25 317 317 355 263 259 269 296 50 50 322 319 362 263 263 270 297 100 356 100 364 Activation due to tyrosine 36 23 30 14 14 12 12 TABLE IV Effect of Phenylalanine and Phenylalanine Plus Asparagine on the Ensymic Hydrolysis of Lintner Soluble Starch Amino Acid, Mg. Cuprous Oxide, Mg. 1 111 1 is o a *fj if II M |f ill OH o A*Pn< OS " S - 2 5 O "5 E O gJ3 g| 8 Exp.l Exp.2 Exp.3 Mg. uto U U-5 Mg. Mg. Mg. 00 280 252 264 100 316 300 100 0.0035 M 0.00003 M 98 0.0035 M 0.00003 M 4 0.00003 M 0.00000018 M 209 100 0.00003 M 0.00000018 M 260 184 100 0.00003 M 0.00003 M 0.00003 M 260 182 Numerous experiments showed that the inactivation of the enzyme by the copper and its reactivation by amino acid were considerably influenced by time and temperature. In Expt. 2 of Table VII the solution stood for 12 minutes before testing; in Expt. 3 it stood for 55 minutes. The influence of temperature is illustrated in Table VIII. TABLE VIII Effect of Temperature on Reactivation of Pancreatic Amylase by Glycine after standing for 20 minutes in 0.00003 M cupric sulphate solution Glycine Temp, of enzyme sol. Cuprous oxide Mg. after 20 min. Mg. 23C 115 100 23 142 12 175 100 12 213 As to the bearing of these experiments with cupric sulphate upon the question whether the role of amino acids is that of a direct accelerator of the enzyme action or rather that of a protector which increases the amount of work done by the enzyme through preventing its deterioration, it cannot be doubted that they establish the pos- sibility of a very marked protective effect without precluding the additional possibility of a more direct action upon the enzyme. Evidently with a low concentration of copper ions in solution these react with amino acid forming copper-amino ions 11 more readily than with the enzyme; and moreover, when the copper ion has already 11 J. T. Barker, Trans. Faraday Soc., 3, 188 (1908). 12 Influence of Certain Amino Acids acted upon and inactivated a part of the enzyme, it apparently may still be taken up by the amino acid, and the enzyme thus freed from the copper may become active again. Does the Amino Acid Act by Retarding the Hydrolytic Destruc- tion of the Enzyme? Another possibility is that the amino acid may act by preventing or retarding the deterioration of the enzyme in its aqueous solution. The very rapid deterioration of water solutions of pancreatic amylase, particularly when highly purified, and the influence of the sodium chloride and secondary phosphate (regularly used to "activate" this enzyme) in retarding the deterioration have been discussed in previous papers from this laboratory. 12 Since the deterioration of enzymic activity, while greatly retarded, is not entirely prevented by the presence of the salts, it is not improbable that the favorable influence of the amino acid may be due at least in part to a further protection of the enzyme from deterioration in the aqueous dispersion in which it acts. In studying this point three methods of investigation have been used. (1) Enzyme solutions with and without amino acid were allowed to stand for a definite length of time at known temperatures in absence of substrate and the resulting loss of activity compared. (2) The effect upon activa- tion due to amino acids of varying the temperature at which the enzyme was allowed to act was determined. (3) A similar series of experiments was carried out in which the length of time of diges- tion as well as the temperature was varied. The method of determining the effect of an amino acid in retarding the deterioration of purified pancreatic amylase in water solution was as follows. 10 mg. of enzyme were dissolved in 100 c.c. of water at ice box temperature containing 5 c.c. M NaCl and 2.5 c.c. M/50 Na 2 HPO 4 . 10 c.c. were pipetted into each of four 25 c.c. beakers, the second of which contained 20 mg. of alanine. At intervals of eight minutes 20 mg. of alanine were added to the third and fourth beakers. None was added to enzyme solution in fourth beaker. These stood at room temperature for 33 minutes after the last addi- tion of alanine. Then 0.6 c.c. from each beaker was pipetted into flasks and substrate poured on and digestions carried out as usual. The substrates digested by the amylase solutions to which the amino acid had been added contained 1.2 mg. alanine put in with 0.6 c.c. 12 Sherman and Schlesinger, Jour. Amer. Chem. Soc. Series of papers (1912-1916). upon the Enzymic Hydrolysis of Starch 13 of enzyme, therefore, to equalize conditions, 1.2 mg. were added to starch dispersions acted upon by the control solution. When tested the solutions to which alanine had not been added were as active as those containing 0.2 per cent of the amino acid. The procedure was then varied, the enzyme being allowed to stand at two different temperatures in solutions containing salt, phosphate and alanine in different combinations. Results are shown in Table IX. In experiments 1 and 2, indicated in following table, the temperature was accurately controlled at 22C., in experiment 3, at 40C. TABLE IX Effect of 0.1 Per Cent Alanine in Retarding Deterioration of Pancreatic Amylase in Solution at 22C and 40C. Alanine NaCl Na 2 HPO 4 Cuprous Oxide, Mg. (0.1%) (optimum cone.) (optimum cone.) Exp. 1 Exp. 2 Exp. 3 22C 22C 40C absent present present 274 144 present present present 275 198 present absent absent 133 absent absent absent 98 absent present absent 273 present present absent 272 absent absent present 238 present absent present 269 The above table indicates that the amino acid does retard the hydrolytic destruction of the amylase. Solutions of pancreatic amylase (containing optimum concentrations of sodium chloride and phosphate) which have stood 1 hour at 40 C. show about y$ greater amylolytic activity when alanine has been added to the solution in advance. Under similar conditions except that the solution is kept at 22C. instead of 40C. there is no measurable difference, probably because the deterioration at this temperature in the presence of optimum concentrations of salts is small in either case. It is probable that at 22C. the amino acid has little or no effect on the rate of hydrolysis of starch by amylase, since, as will be shown later, the activation decreases markedly with temperature. It further appears from Table IX that amylase solutions to which the usual NaCl and Na 2 HPO 4 have not been added are protected by alanine even at 22. At this temperature the amino acid is apparently capable of replacing either the sodium chloride or the phosphate without altering the 14 Influence of Certain Amino Acids activity of the solution, but not both salts. In these experiments, however, the conditions were such as to result in greater deterioration than occurs in our ordinary tests of enzyme activity both because of longer exposure of the enzyme to water and because of the absence of its substrate. If protection against hydrolytic destruction is a partial explanation of the increased activity of enzymes in the presence of amino acids, it would be logical to expect that any condition favoring the hydrolysis of the enzyme molecule, such as a higher temperature or subjection to a given temperature for a longer period, would cause greater ap- parent activation by the amino acid. To test this point, a series of experiments was planned in which 30 minute and 60 minute digestions by pancreatic amylase with and without amino acid were carried out at temperatures ranging from 30-75C. Table X shows the results obtained with glycine at different temperatures when 0.6 c.c. of 0.01 per cent enzyme solution acts upon 100 c.c. of substrate for 30 minutes and when one-half the amount acts upon the same amount of substrate for 60 minutes. The experiments were repeated with phenylalanine replacing glycine, the results of which are given in Table XI. The purified pancreatic amylase preparation employed in the phenylalanine experiments was about a year older than that used with glycine and only two-thirds as active, consequently, one- third more of the solution was added to each digestion mixture. TABLE X Effect of Variation of Temperature on Activation of Purified Pancreatic Amylase by Glycine 30 minute digestions 60 minute digestions Cuprous Oxide, Mg. Cuprous Oxide, Mg. 30C 40 50 55 57 60 65 66.5 70 75 "ei Activation B|| "bo Activation ^ & o due to 50 mg. o due to 50 mg. "3>- fc glycine 10 "So o. glycine 155 136 19 14% 142 114 28 24% 252 220 32 14% 287 248 39 12% 366 311 55 17% 343 259 84 32% 393 291 102 35% 369 214 155 72% 363 251 112 45% 367 167 200 120% 380 183 197 107% 320 84 236 281% 176 58 118 203% 99 24 75 312% 110 30 80 266% 52 19 33 173% 55 24 31 129% 18 8 10 125% 0% 0% upon the Enzymic Hydrolysis of Starch 15 TABLE XI Effect of Variation of Temperature on Activation of Purified Pancreatic Amylase by Phenylalanine 30 minute dijjest'ons 60 minute digestions Cuprous Oxide, Mg. Cuprous Oxide, Mg. Ill o-S Activation a> C C ""* S..S Activation s8 o a due to 50 mg. j S o c due to 50 me. -S"3 m o< rt ^ ^ phenylalanine S'S'3 ^"a phenylalanine 183 164 19 12% 173 143 30 21% 291 258 33 12% 290 247 43 17% 340 255 85 33% 300 194 106 55% 291 181 110 60% 226 94 132 140% 134 66 68 103% 105 37 68 184% 42 25 17 68% 22 12 10 83% 10 7 3 43% 6 4 2 50% H 30 C 40 50 55 60 65 70 The results of the above experiments afford striking evidence that deterioration of the enzyme with increase in temperature is retarded by amino acids. For the 30 minute digestions, beginning with an increase at 30C. of 19 mg. of cuprous oxide or 14 per cent, the activating effect of glycine reaches a maximum of 197 mg. of cuprous oxide or over 100 per cent at 60C. Above this temperature the acceleration as represented by increase in mg. of cuprous oxide declines sharply, although the percentage activation continues to in- crease up to 66.5C. The rapid falling off in activity after 60C. is doubtless due to coagulation of the amylase which is not prevented by the amino acid. The experiments with phenylalanine show the same general effect though not as marked as in the case of glycine. Maximum activation occurs at 55C. instead of 60C. after which the decline is rapid. It will be observed from the tables that the two amylase preparations in absence of either amino acid behave quite differently, the less active one, employed in connection with phenylalanine, being destroyed more rapidly with increasing temperature. Glycine added to a sub- strate hydrolyzed by this enzyme at 60C. gave practically the same result as obtained with phenylalanine. Therefore, it appears that the lack of agreement in the results is due not to dissimilarity in the action of the two amino acids but rather to some difference in the amylases, probably connected with the deterioration which had already occurred in the less active preparation. 16 Influence of Certain Amino. Acids The most evident explanation of this marked temperature effect is that the amino acids preserve the enzyme in solution from the destructive influence of heat. In so far as the result of enzymic hydrolysis is concerned, increase in temperature exerts two opposite influences upon amylolytic action. It accelerates the velocity of conversion of starch into sugar and at the same time increases the rate of deterioration of the enzyme. The second reaction being retarded by the presence of one of the decomposition products, the first effect, that is, increase in the rate of hydrolysis of the starch, becomes more noticeable. When hydrolysis continued for 60 minutes the amino acids pro- duced a greater apparent activation at all temperatures until after coagulation of the enzyme had occurred than was observed for the shorter period digestions. Digestions carried out at 40 C. for periods of time from 20 minutes to 3 hours with and without glycine and tyrosine show the same increase in apparent activation with length of time of action of the amylase. This is what would be expected if the amino acid activates by protecting against deterioration, since the longer the enzyme is subjected to an injurious temperature, the greater the deterioration and consequently the more marked the activating effect in digestion mixtures in which the destruction is partially prevented. The fact, demonstrated by the above data, that the presence of certain amino acids retards the deterioration of the enzyme consti- tutes an interesting addition to the evidence supporting the view that the enzyme itself is a substance of protein nature or which contains protein as an essential constituent. SUMMARY Addition of glycine, alanine, phenylalanine or tyrosine caused an undoubted increase in the rate of hydrolysis of starch by purified pancreatic amylase, commercial pancreatin, saliva or purified malt amylase. Less marked results were obtained with the less sensitive enzyme materials, malt extract, takadiastase and aspergillus amylase. Each of the four amino acids here studied as well as aspartic acid and asparagine previously investigated, showed a similar favorable influence upon the enzymic hydrolysis of starch. upon the Enzymic Hydrolysis of Starch 17 The addition of a mixture of two amino acids produced no greater effect than would result from the same concentration of one of them. In these experiments the favorable effect of the added amino acid was not due to any influence upon hydrogen ion concentration nor to combination of the amino acid with the products of the enzyme reaction. The addition of one of these amino acids is a very effective means of protecting the enzyme from the deleterious influence of cupric sulphate and may even serve to restore to full activity an enzyme which has been partially inactivated by copper. The favorable influence of the amino acid is evidently due in part at least to a protection of the enzyme from deterioration in the aqueous solution in which it acts. This view is supported by the following facts: (1) Solutions of pancreatic amylase (containing optimum concentrations of chloride and phosphate) which have stood 1 hour at 40 C. show ^ greater activity when alanine has been added to the solution in advance. Amylase solutions to which the chloride and phosphate have not been added are protected by alanine at 22 C. (2) There is a striking increase in activation by glycine and phenyl- alanine with increased temperature until coagulation of the enzyme occurs. (3) At the same temperature, there is greater apparent activation when hydrolysis is allowed to proceed for 1 hour than when the action is stopped at the end of 30 minutes. This explanation of the mode of action of amino acid does not preclude the possibility of a more direct influence upon the activity of the enzyme. VITA Florence Walker was born at Oriole, Md., October 15, 1887. She was prepared for college in the Blackstone School for Girls, Black- stone, Va. Entering Randolph-Macon Woman's College in 1905 she received the degree of Bachelor of Arts in 1909. Until 1917 she was Instructor in Chemistry in Randolph-Macon. During the years 1917-18 and 1921-22 and Summer Sessions 1916 and 1917 she pursued the study of chemistry in Columbia University, receiving the degree of Master of Arts in 1918. From 1918 to 1921 she was Carnegie Research Assistant to Professor H. C. Sherman, during which period she was co-author with Professor Sherman of several papers on amylases. For the past year she has held a University Fellowship in Chemistry. TX.A, BELow 11 " 1831 DAT E BIOLOGY LIBRARY" A78719