A STUDY OF THE CONCENTRATION AND PROPERTIES OF TWO AMYLASES OF BARLEY MALT

A STUDY OF THE CONCENTRATION AND PROPERTIES OF TWO AMYLASES OF BARLEY MALT BY M. L. CALDWELL AND S. E. DOEBBELING (From the Department of Chemistry, Columbia University, New York) (Received for publication, June 14, 1935) The experiments discussed briefly here give additional evidence to that previously reported (l-6) for the presence of two distinct amylases in extracts of barley malt by showing that they may be simultaneously concentrated in different fractions obtained from the same extracts without appreciable losses of activity of either at any given stage. The preparations obtained are similar in certain respects to the cr- and P-amylases which have been reported by other investigators such as Kuhn (7) and Holmberg (6) but differ in other respects. Because of this finding, and the confusion in the literature over the terminology (1, 2, 7), we refrain at this time from the use of these terms. The preparations discussed here appear to be more highly purified than any previously reported and do not exhibit the differences, either in conditions which favor their action or in stability upon heating in aqueous solution, which have been reported for less highly purified products (2, 5, 6). The data so far obtained also do not indicate the necessity for the presence of unknown activating agents, so called kinases (5,8). EXPERIMENTAL Fractionation-The method for obtaining one type of preparation from extracts of barley malt has already been described (9). It consists of repeated fractionations by ammonium sulfate with intervening dialyses, followed by fractional precipitation with alcohol. The final products of the process have exceedingly high activity as judged by the weight of maltose formed from starch in 30 1 Barley malt of high diastatic activity was kindly supplied by Messrs. A. Schwill and Company through the courtesy of Mr. Robert Schwartz and Mr. Oscar Ruh. 739

Arnylases of Barley Malt minutes at 40 @O-12), but only negligible activity when judged by the weight of starch hydrolyzed to products which give no blue color with iodine, as determined by a modification (13) of the Wohlgemuth method (14). Thus, of two types of amylase activity found in the original malt extracts, one is markedly concentrated in, while the other is practically eliminated from these preparations. Renewed study of the purification process, with examination of each fraction for both kinds of activity, showed that the amyloelastic activity tends to be concentrated in the earlier stages of the refractionation with ammonium sulfate and in the precipitates formed by the lower rather than the higher concentrations of alcohol. These fractions, which had previously been discarded because their activity was low when judged by the weight of maltose formed from starch in 30 minutes at 40 (12), are now found to represent lo- to 30-fold concentrations of the amyloclastic activity (13) of the malt extracts. Moreover, quantitative measurements of both types of activity, based upon the total volumes and solids of the solutions, show no appreciable losses of either activity at any given stage in the process. This constitutes strong evidence that the two types of products represent the concentration of two distinct amylases and not merely manifestations of different properties of the same enzyme under different treatments or conditions. Activities-In studying the purified products, the usual 30 minute measurements of activity (12) already referred to, which were used to follow the fractionation of the amylases, were supplemented by quantitative comparisons of their action throughout the course of the hydrolysis of starch. These were carried out as follows : 2 per cent starch, adjusted to ph 4.5 and 0.01 M acetate (la), was brought to 40 and poured upon the enzyme solution in a flask. After being rotated gently to insure thorough mixing, the flask was placed in a thermostat regulated to 40 and kept there throughout the reaction. At definite time intervals, 10 cc. portions of the hydrolysis mixture were withdrawn and delivered into a flask which was immediately plunged into rapidly boiling water and kept there for exactly 2 minutes. After rapid cooling under running water

M. L. Caldwell and 8. E. Doebbeling 741 and dilution with 90 cc. of distilled water, the reducing action was determined by a Fchling s gravim.etric method (10, 12). As nearly simultaneously as possible with the removal of the 10 cc. portions for the saccharogenic measurements (immediately before and immediately after), 1 cc. aliquobs of the hydrolysis mixture were withdrawn for amyloclastic activity measurements ED WITH IODINE 0 30 60 90 120 150 180 TIME: IN MINUTES FIG. 1. Typical data showing the earlier stages of 6he course of the hydrolysis of starch when catalyzed by two amylases of barley malt. Activity is expressed as maltose formed from 2 per cent starch in the presence of 0.01 M acetate at ph 4.5 and 40. Curve 1, preparation of malt amylase typical of the highly active final products of the purification process; Curve 2, preparation of malt amylase typical of those with a preponderance of high initial amyloclastic activity. and delivered into test-tubes. After dilution with approximately 10 cc. of water, 1 drop of 0.1 N iodine in potassium iodide was added from a dropping bottle and the resulting colors compared with the Milton Bradley color standards (15). Aliquots for measurements of both types of activity were taken rapidly during the stages when the color with iodine was changing from blue to red.

742 Amylases of Barley Malt As the main interest centered in the earlier stages of the reactions, no toluene was a,dded to these solutions. Precautions were taken to minimize evaporation at all stages. Typical data showing the course of the hydrolysis of starch when catalyzed by the two types of amylase preparations are summarized in Fig. 1. Here, the reducing values of the hydrolysis mixtures, expressed as maltose, are plotted against time. The data are comparable, as the starch was hydrolyzed under exactly the same conditions in both cases, and, furthermore, the proportions of starch and enzyme were so adjusted that the same weight of maltose was formed in the same time at some point in the hydrolysis (118 mg. of maltose in 78 minutes). The reaction mixtures were both adjusted to ph 4.5 which had been found to permit optimal activity of both amylases under the conditions used ((12) ; also later section of this report). The data summarized in Fig. 1 show that the two amylases catalyze the hydrolysis of starch in a different manner. In one case (Curve l), maltose is formed rapidly at first but soon approaches a maximum and then increases only gradually. In the other case (Curve 2), maltose appears more slowly at first but its formation is more sustained. Beyond the time at which the same weight of maltose was formed in the two reaction mixtures, higher values for maltose were obtained with the amylase represented in Curve 2 than with that represented in Curve 1. This makes it evident that the relative saccharogenic activities of the two amylases will depend upon the time interval in the hydrolysis chosen for the comparison. The relation between maltose production and color with iodine in the two types of reaction mixtures is also instructive. The rapid formation of maltose in the early stages of the hydrolyses represented by Curve 1 (Fig. 1) is accompanied by a lagging of products which give a blue color with iodine, which, if taken alone, would give a misleadingly low impression of the activity of the amylase. On the other hand, the slower production of maltose in hydrolyses catalyzed by the amylase represented in Curve 2 is accompanied by the rapid disappearance of products which give a blue color with iodine. Some typical data follow. With the reaction mixture represented by Curve 1, the following results were obtained: blue with iodine at 30 minutes and 106 mg.

M. L. Caldwell and S. E. Doebbeling 743 of maltose per 10 cc.; approaching the red end-point with iodine at 1300 minutes and 154 mg. of maltose per 10 cc.; no color with iodine at 2700 minutes and 187 mg. of maltose per 10 cc., which is approximately 89 per cent of the theoretical yield* of maltose. With the reaction mixture represented by Curve 2, the following results were obtained: clear red color with iodine in 30 minutes and 65 mg. of maltose per 10 cc.; no color with iodine in 45 minutes and 85 mg. of maltose per 10 cc.; 209 mg. of maltose per 10 cc., in 1300 minutes. This represents the theoretical yield2 of maltose. A further comparison at the stage at which the two reaction mixtures just failed to give a blue color with iodine showed that the preparation represented by Curve 2 had hydrolyzed 1000 times its weight of starch and formed 337 times its weight of maltose in 30 minutes at 40 at a dilution of 1 part to 51,000; while the preparation represented by Curve 1 had hydrolyzed 6349 times its weight of starch and formed 4894 times its weight of maltose at a dilution of 1 part to 318,250 but had required 1300 instead of 30 minutes to reach this stage. It is evident that these typical preparations represent the concentration of two different amylases. Apparently, also, the former has not yet been as completely separated from inert material as the latter. For this reason, the det,ails of the fractionation are omitted at this time. InJluence of Hydrogen Ion Activity-Several investigators, including Ohlsson (2) and Holmberg (6), have reported that the hydrogen ion activity which affords optimal amylase action differs for the two amylases of barley malt. The values found best by these and other investigators are, however, not in agreement (11, 12, 16-18) and may not be comparable as they were obtained under different conditions and in the presence of different buffers. A brief summary is given here of a study of this point, made under conditions which differed only in the hydrogen ion activities of the reaction mixtures and in the use of preparations of malt amylase of different degrees of purification and separation from each other. 2 per cent starch containing a total concentration of 0.01 M acetate ion (12) was used in all cases. The starch was adjusted to 2 The weight of the starch multiplied by 1.05 is taken as the theoretical value of maltose.

744 Amylases of Barley Malt different ph values by changes in the proportions of equimolar acetic acid and sodium acetate. The ph values included ph 4.5 which had been found (12) to permit optimal action of the amylase with the high initial saccharogenic activity and ph 5.3 which is representative of the ph values found favorable by other investi- I I I I I I I I I I I I I 38 42 4.6 51) 54 58 62 PH FIG. 2. Typical data showing influence of hydrogen ion activity upon the saccharogenic activities of highly purified preparations of two amylases of barley malt. Activity is expressed as maltose formed from 2 per cent starch in the presence of 0.01 M acetate in 30 minutes at 40. Curve 1, malt amylase preparation with preponderance of high initial saccharogenic activity; Curve 2, malt amylase preparation with preponderance of high initial amyloclastic activity. gators for the malt amylase which exerts the high initial amyloelastic activity. Comparable measurements of the so called saccharogenic and amyloclastic activities of the amylases were made at a given time interval in the usual manner (12, 13) in which the amylases reacted with equal portions of the starch at different ph values for periods of 30 minutes at 40. The influence of hydrogen ion

M. I,. Caldwell and S. E. Doebbeling 745 activity was aiso studied throughout the course of the hydrolysis of starch as catalyzed by the two amylases. In this case, the reaction mixtures were held at 40 for several hours and measurements made at frequent intervals by removing aliquots and measuring them for both types of activity as described above. Fig. 2 summarizes typical data for the influence of hydrogen ion activity upon the saccharogenic activities of two highly purified preparations of malt amylase which show a marked preponderance of one or the other type of amylase activity. Curve 1 represents data for a preparation with high initial saccharogenic and low initial amyloclastic activity, while Curve 2 represents data for a preparation with low initial saccharogenic and high initial amyloclastic activity. It is seen that in both cases the saccharogenic activity is favored by hydrogen ion activities corresponding to ph values of 4.3 to 4.6. The preparations used in this work probably do not represent a complete separation of the two amylases. This ideal appears t.o be more nearly approached in the products with a preponderance of the high initial saccharogenic activity than in those in which the high initial amyloclastic activity predominates. For this reason, the saccharogenic activity measured as described above is probably, at least in the latter case, due to the action of both amylases. Even if this objection is accepted, the data given in Fig. 2 definitely disprove the idea that the saccharogenic activities of these two amylases are favored by different hydrogen ion activities. The amyloclastic action (13) of the amylase with high initial amyloclastic activity is also favored by hydrogen ion activities corresponding to ph values of 4.3 to 4.7. This is shown by the following t,ypical data in which the micrograms of enzyme preparation necessary to cause the hydrolysis, to products which give no blue color with iodine, of 5 cc. of 1 per cent starch in 30 minutes at 40 are given with the corresponding ph values: 14.2 micrograms at ph 4.2, 10.6 micrograms at ph 4.3, 4.4, 4.5, 4.6, and 4.7, 12.0 micrograms at ph 4.9, and 13.0 micrograms at ph 5.2. Similar measurements were not feasible with the preparations of high initial saccharogenic activity, as the action thus studied is negligible in these products. Data for the influence of hydrogen ion activity upon t,he course

746 bylaws of Barley Mdt of the hydrolg?sis of starch as ca.talyzed by the amylases of malt are omitted for the sake of brevity but show conclusively that ph4.5 is also more favorable to the action of both amylases throughout the entire reaction than ph 5.3 which had been recommended by some previous investigators. Stabilities-The results of an extensive series of experiments with amylase solutions of different degrees of purification show no measurable differences (2) in the stabilities of the two amylases of barley malt upon being heated in aqueous solution after partial purification. The extent of the losses is in both cases influenced by such factors as accompanying impurities, degree of dilution, and the time and temperature of heating. The following results are typical. When a dilute extract of barley malt (40 mg. of solid per 100 cc.) was held at 70 for 10 minutes (2), it lost 90 per cent of its saccharogenic and 70 per cent of its amyloclastic activities. More concent,rated extracts, similarly treated, showed no measurable activity of either kind, probably due to a carrying down of remaining active material with coagulated proteins. A solution which contained 0.05 mg. per cc. of a preparation which exhibited approximately 30-fold concentration of both activities over those of the original malt extract gave no evidence of either kind of activity after being held at 70 for 2 minutes. When this solution was held at lower temperatures for definite time intervals no measurable differences in the losses of the two activities were observed. SUMMARY AND CONCLUSIONS A method has been developed for the separation and simultaneous concentration of two amylases in different fractions obtained from the same extracts of barley malt without appreciable losses of either type of activity at any given stage. This constitutes additional and direct evidence for the presence of two distinct amylases in such extracts. After purification, both amylases rapidly lose their activities upon being heated in aqueous solutions and exhibit no marked differences in this property. Both types of highly purified products are free from carbohydrate but give positive protein color reactions. Comparative studies of the course of the hydrolysis of starch

M. L. Caldwell and S. E. Doebbeling 747 by the two types of purified products make it evident that the amylases catalyze the hydrolysis of amyloses in a different manner. In the presence of 0.01 M acetate and in measurements at 40, hydrogen ion activities corresponding to ph values of 4.3 to 4.6 favor the saccharogenic action of both amylases of barley malt. Under similar conditions, the amyloclastic action of the amylase with a preponderance of this activity is also favored by hydrogen ion activities corresponding to ph 4.3 to 4.7. The above hydrogen ion activities favor the action of both amylases throughout the course of the hydrolysis of starch as well as in measurements of relatively short duration. BIBLIOGRAPHY 1. Syniewski, V., Biochem. Z., 158,87 (1925); 263,266 (1932). 2. Ohlsson, E., Compt.-rend. trav. Lab. Carlsberg, 16, No. 7 (1926); Z. physiol. Chem., 189,17 (1930). 3. Nordh, G., and Ohlsson, E., 2. physiol. Chem., 204, 89 (1931-32). 4. van Klinkenberg, G., Z. physiol. Chem., 209, 253 (1932). 5. Waldschmidt-Leitz, E., Reichel, M., and Purr, A., Naturwissenschajten, 20, 254 (1932). 6. Holmberg, O., Biochem. Z., 268, 134 (1933); 266, 203 (1933); Ark. Kemi, Mineral. o. Geol., 11 B, No. 4, 6 (1932). 7. Kuhn, R., Ann. Chem., 443, 1 (1925). 8. Waldschmidt-Leitz, E., and Purr, A., Z. physiol. Chem., 203, 117 (1931); 213,63 (1932). 9. Sherman, H. C., Caldwell, M. L., and Doebbeling, S. E., J. Biol. Chem., 104, 501 (1934). 10. Sherman, H. C., Kendall, E. C., and Clark, E. D., J. Am. Chem. SOL, 32, 1073 (1910). 11. Sherman, H. C., Thomas, A. W., and Baldwin, M. E., J. Am. Chem. Sot., 41, 231 (1919). 12. Sherman, H. C., Caldwell, M. L., and Boynton, H. H., J. Am. Chem. Sot., 62, 1669 (1930). 13. Sherman, H. C., and Thomas, A. W., J. Am. Chem. Sot., 37,623 (1915). 14. Wohlgemuth, J., Biochem. Z., 9, 1 (1908). 15. Mulliken, S. P., A method for the identification of pure organic compounds, New York (1922). 16. Pringsheim, H., and Leibowitz, J., Biochem. Z., 161,456 (1925). 17. Samec, M., and Waldschmidt-Leitz, E., Z. ph,ysioz. Chem., 203,16 (1931). 18. Pronin, S., Biochem. Z., 249,7 (1932).

A STUDY OF THE CONCENTRATION AND PROPERTIES OF TWO AMYLASES OF BARLEY MALT M. L. Caldwell and S. E. Doebbeling J. Biol. Chem. 1935, 110:739-747. Access the most updated version of this article at http://www.jbc.org/content/110/3/739.citation Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's e-mail alerts This article cites 0 references, 0 of which can be accessed free at http://www.jbc.org/content/110/3/739.citation.full.h tml#ref-list-1