THE EFFECTS OF DIFFERENT BUFFERS ON THE ACTIVITY OF [PDF]

THE

EFFECTS OF DIFFERENT BUFFERS ACTIVITY OF &AMYLASE BY

GERALD

(From the Department

A. BALLOU

AND

J. MURRAY

Stanford

of Chemistry,

(Received for publication,

January

ON THE LUCK

University,

California)

14, 1941)

EXPERIMENTAL

Preparation of Bu$ers-The proportions of buffer acid and sodium hydroxide to be mixed to obtain a desired pH and ionic 233

Downloaded from http://www.jbc.org/ by guest on April 2, 2019

In a previous investigation by Ballou and Luck (l), the specific effects of different buffers on the pH optimum of taka-diastase activity and the relative activities of the enzyme at the pH optima and on the acid and alkaline sides of the optima were studied. The present work constitutes an extension of this investigation to /3-amylase. The following ten buffers, in systems of constant ionic strength, were used: formate, acetate, propionate, butyrate, valerate, phenyl acetate, phthalate, succinate, phosphate, and In addition, the activity-pH relationship for a valerate citrate. buffer was determined, in which a constant total valerate concentration of 0.07 M was maintained. This was done to permit comparison with the results obtained at constant ionic strength over the same pH range. Because of the pronounced effects of concentrated urea solutions on proteins, it was thought that it would be of interest to include in this work several runs in the presence of concentrated urea. Gerber (2) reported that, while low concentrations of urea had little effect on diastatic activity, there was marked retardation in higher concentrations. A concentration of 0.133 M urea was found to have a slight inhibitory influence on malt amylase at pH 4.5 by Filipowicz (3). In contrast with these results on amylase, several investigators (4-7) have reported that high concentrations of urea promote the action of the proteolytic enzymes, pepsin, papain, asclepain, and trypsin.

234

,&Amylase Activity

Downloaded from http://www.jbc.org/ by guest on April 2, 2019

strength were determined as described in the report on takadiastase (1). Equations 3 and 4 of this previous work were employed in the present investigation to calculate the correct proportions of sodium hydroxide and potassium dihydrogen phosphate for the phosphate buffer mixtures; some phosphoric acid was added to obtain pH values lower than 4.5, in which region very little buffering occurred. For the valerate buffer mixtures of constant total valerate concentration (0.07 M), 0.76 cc. of the anhydrous acid was mixed with variable amounts of 1.0 N NaOH and diluted to 100 cc. Preparation of Substrate-Bu$er Mixtures-The substrate-buffer mixtures were prepared in an identical manner to that reported in the work on taka-diastase (l), except that a final starch concentration of 1 gm. of dry starch per 100 cc. of solution was used. The 2 M urea mixtures were prepared in the following manner. To a 100 cc. volumetric flask, calibrated for delivery, were added the desired volume of glacial acetic acid and 5 cc. of 1.0 N NaOH, 50 cc. of starch solution containing 1 gm. of dry starch, 25 cc. of concentrated urea solution containing 12 gm. of urea, and sufficient redistilled water to bring the final volume to 100 cc. In the preparation of the 4 M urea mixtures, 40 cc. of starch solution containing 1 gm. of starch and 50 cc. of urea solution containing 24 gm. of urea were pipetted into the 100 cc. volumetric flask. Preparation of P-Amylase-Several investigators (8-11) consider whole wheat flour an excellent source of /3-amylase, and report that hard winter wheat is in most respects superior to barley. The following modified procedure for the isolation of P-amylase includes some steps that were suggested by the papers of Hanes and Cattle (12), van Klinkenberg (lo), and Myrback and &tenblad (13). 100 gm. of wheat flour, prepared with a hand grist mill, were stirred slowly and thoroughly with 300 cc. of distilled water at room temperature for fr hour. The thick suspension was then centrifuged to separate the coarser material. The centrifugate, a cloudy suspension of fine particles, was again centrifuged in cellulose nitrate tubes in an angle centrifuge for about 20 minutes at 4000 R.P.M. at a temperature of O-5”. The centrifugate (about 200 to 210 cc.) still had a slightly turbid appearance. The remaining steps in the procedure were carried out entirely in the cold room.

G. A. Ballou and J. M. Luck

Downloaded from http://www.jbc.org/ by guest on April 2, 2019

An equal volume of cold, redistilled, 95 per cent ethyl alcohol was added to the chilled centrifugate with stirring. Slow stirring was continued for several minutes after the addition of the alcohol. The best results were obtained when both alcohol and centrifugate were chilled thoroughly before mixing. During 15 minutes of standing a flocculent precipitate was brought down by the alcohol. This was centrifuged off with the angle centrifuge and discarded. To the clear centrifugate (about 385 cc.) 1.8 times its volume of This cold 95 per cent alcohol was added slowly with stirring. gave a final alcohol content of approximately 80 per cent by volume. The mixture was stirred slowly for several minutes and allowed to stand for 5 to 10 minutes, during which time a white flocculent precipitate settled out. The fraction coming down between 50 and 80 per cent alcohol concentration was considered to contain most of the fl-amylase (10, 12, 13), and was centrifuged off. The rather well packed sediment in the centrifuge tubes was transferred to a small evaporating dish, broken up into smaller particles, and desiccated in a high vacuum over phosphorus pentoxide, after removal of most of the alcohol by continuous pumping for from 1 to 2 hours. A creamy white, caked material was obtained after it had remained in the evacuated desiccator overnight. The material was pulverized and stored in a screw top bottle in the cold room. Approximately 0.9 gm. was obtained per 100 gm. of whole wheat flour. The enzyme powder dissolved readily in water and left only traces of insoluble material which settled to the bottom of the solution on standing for a few minutes. Its activity compared favorably with that reported by the investigators mentioned above (10, 12, 13), and on the whole this process was less complicated and more rapid. Several gm. of the material were prepared in a day. 1 mg. of the enzyme powder was found to produce, at 30” and in the presence of an acetate buffer at pH 5.0, 6.8 mg. of maltose per minute from 100 cc. of 1 per cent starch solution, and 7.25 mg. per minute from a 2 per cent starch solution. Preparation of Reaction Mixture-25 mg. of /3-amylase powder were dissolved in several cc. of redistilled water and diluted to 25 cc. For each run 1 cc. of this enzyme solution was pipetted into a 250 cc. Florence flask, and the digestion carried out at 30” in a manner similar to that described in the work with taka-diastase (1).

236

@Amylase Activity

A modified Willstatter-Schudel hypoiodite method (14, 15) was employed to follow the increase in reducing power of the digest. In several control experiments it was shown that the presence of 2 M urea did not interfere with the stoichiometric oxidation of the carbohydrate reducing groups by the hypoiodite reagent. Results

82.2 I 3 kj 1.8 I

0

FORMAT.5 ACETATE 0 PROPIONATE 6 BUTYRATE

A g IA

A SUCCINATE 0 PHENYLACETATE X PHTHALATE

FIG. 1 FIG. 2 1 AND 2. Activity-p*1 curves for P-amylase with the reciprocal the time for the reduction of 2 cc. of 0.05 N 1~ as the index of activity. FIGS.

of

per cent of the theoretical maltose) was accepted as the index of enzyme activity. Table I presents a list of the buffers and the corresponding pH optima for p-amylase and taka-diastase (1) at a constant ionic strength of 0.05. In Fig. 1 the pH optimum shifts slightly to the alkaline side, and the relative activity at the optimum diminishes gradually as one ascendsthe homologous series from formate to valerate. On the alkaline side of the pH optima the activity curves for the monobasic acid buffers coincide, and lie slightly under those for the polybasic acid buffers. On the acid side of the optima, however, the saccharogenic activity of P-amylase varies appreciably with the buffer. Increase of carbon chain length of the buffer acid

Downloaded from http://www.jbc.org/ by guest on April 2, 2019

Fig. 1 presents the activity-pH curves for P-amylase in the presence of formate, acetate, propionate, butyrate, and valerate buffers, and Fig. 2 the corresponding curves for phenyl acetate, phthalate, succinate, citrate, and phosphate buffers. The reciprocal of the time for the reduction of 2 cc. of 0.05 N IZ by a 5 cc. aliquot of digest (equivalent to the production of about 26

G. A. Ballou and J. M. Luck exhibits a greater inhibitory influence diastase (1) in the acid region. The curves for phosphate and citrate relative activity of the enzyme at the the two aromatic buffers is less than The zones of optimum pH activity for TABLE

Summary

of pH

Optimajor

p-Amylase Di$erent

FIG. 3. Activity-pH

curves

for

practically coincide. The optimum in the presence of that for the other buffers. p-amylase in the fatty acid

I and Taka-Diastase Buffers

in Presence

pH optimum for o-amylase

pH optimum for taka-dia.&am

4.7 4.8 5.0 5.0 5.2 5.0 5.0 5.3 4.5-4.9 4.5-4.8

4.6-5.2 5.1 5.1 5.1 5.1 5.1 5.1-5.3 5.4-5.6 5.1-5.3

Formate ............................ Acetate ............................ Propionate ......................... Butyrate ........................... Valerate ............................ Phenyl acetate ..................... Succinate .......................... Phthalate .......................... Citrate ............................. Phosphate. .........................

0

than on taka-

CONSTANT

p-amylase

of

TO

in the

presence

of a valerate

buffer.

buffers are narrower than for taka-diastase, and slightly removed toward the acid side. Fig. 3 presents two markedly different activity-pH curves for p-amylase with a valerate buffer. The important observation in Fig. 3 is the wide deviation of the curves on the acid side of the optimum. Over the pH range of one curve, a constant ionic

Downloaded from http://www.jbc.org/ by guest on April 2, 2019

BUffer

on p-amylase

237

,&Amylase Activity

Downloaded from http://www.jbc.org/ by guest on April 2, 2019

strength of 0.05 was maintained, while the other curve was constructed for a buffer of total, constant valerate concentration (0.07 M). The concentration of 0.07 M valerate was chosen because it was the same as the total valerate concentration at the pH optimum of the activity at constant ionic strength. Wisansky (16) published two similar curves for invertase in an acetate buffer. That a specific buffer influence on the shape of the activity-pH curve exists is evident from the results obtained in this investigation with /3-amylase, and from those of the previous study on takadiastase (1). It can also be observed that the concentrations of the buffer constituents exert an appreciable effect. From the standpoint of an electrostatic association of the protein ion and the oppositely charged buffer ions, and on the assumption that the pH optimum coincides with the isoelectric point (17), it is not difficult to understand the relatively small influence of the variable buffer anions on the optimum pH. The observed shifts, however, may be accounted for by a reasoning somewhat similar to that advanced by Adair and Adair (18) and Tiselius and Svensson (19) ; namely, that near the isoelectric point the protein combines to some extent with the buffer ions, preferentially the anions. This argument assumes implicitly that /3-amylase and taka-diastase are proteins. On the alkaline side of the pH optima, where the amphoteric enzyme molecule is increasingly negative in charge, the buffer cation consists only of sodium ion for eight of the buffers, and of a mixture of sodium and potassium ions for the phthalate and phosphate buffers. Hence, a coincidence of the activity curves is to be found in this region. On the acid side of the pH optima, it may be expected that specific influences would be in evidence by virtue of the differences in nature and size of the various oppositely charged buffer anions. The possibility of a specific action of the neutral buffer acid on the activity of the enzyme must also be considered. A decrease in pH for a given buffer necessarily involves an increasingly neutral acid concentration. It is possible that the relatively higher neutral acid concentration at lower pH values fosters an increasing rate of inactivation of the enzyme (and specifically so) on standing in solution; or it may be that the neutral acid molecules are adsorbed by the protein ions and thus interfere with their enzymic properties.

239

G. A. Ballou and J. M. Luck

In Fig. 4 two activity-pH curves for P-amylase and an acetate buffer are presented, the lower one resulting from experiments in the presence of 2 M urea. Owing to the greatly reduced activity in the presence of urea, it was found expedient to use as the index of activity the reciprocal of the time necessary for the reduction of 1.40 cc. of 0.05 N 1~ by a 5 cc. aliquot of digest. Although the

of no urea

curves for fi-amylase and 2 M urea.

FIG. 5. Time mum

curves for P-amylase in the presence of no urea, 2

M

with

an acetate

and an acetate buffer urea, and 4 M urea.

buffer

in the

at the pH

opti-

activity was reduced by the urea, the curves remained approximately parallel, with practically no shift in the pH optimum. Three time curves are plotted in Fig. 5 which illustrate the relative activity of fi-amylase at its pH optimum in the presence of no urea, 2 M urea, and 4 M urea. The number of cc. of 0.05 N 1~reduced by a 5 cc. aliquot of digest is plotted against the time in minutes. These results agree with the findings of Gerber (2) and Filipowicz (3).

Downloaded from http://www.jbc.org/ by guest on April 2, 2019

FIG. 4. Activity-pH presence

240

@Amylase Activity SUMMARY

BIBLIOGRAPHY

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

Ballou, G. A., and Luck, J. M., J. Biol. Chem., 136, 111 (1940). Gerber, C., Corn@. rend. Xoc. biol., 71, 41 (1911). Filipowicz, B., Biochem. J., 26, 1874 (1931). Steinhardt, J., J. Biol. Chem., 123, 543 (1938). Winnick, T., Davis, A. R., and Greenberg, D. M., J. Gen. Physiol., 23, 275 (1940). Crippa, G. B., Maffei, S., and Wylde, E., Gazz. chim. ital., 69,731 (1939). Anson, M. L., J. Gen. Physiol., 22,79 (1938). Stamberg, 0. E., and Bailey, C. H., J. BioZ. Chem., 126, 479 (1938). Sandstedt, R. M., Kneen, E., and Blish, M. J., Cereal Chem., 16, 712 (1939). van Klinkenberg, G. A., 2. physiol. Chem., 209, 253 (1932). Bailey, C. H., The chemistry of wheat flour, American Chemical Society monograph series, New York (1925). Hanes, C. S., and Cattle, M., Proc. Roy. Sot. London, Series B, 126, 387 (1938). Myrbiick, K., and tjrtenblad, B., 2. physiol. Chem., 293, 107 (1937). Willstltter, R., and Schudel, G., Ber. them. Ges., 61,780 (1918). Blom, J., Bak, A., and Braae, B., 2. physiol. Chem., 260, 104 (1937). Wisansky, W. A., J. Am. Chem. Sot., 61, 3330 (1939). Sherman, H. C., Thomas, A. W., and Caldwell, M. L., &. Am. Chem. Sot., 46, 1711 (1924). Adair, G. S., and Adair, M. E., Biochem. J., 26, 1230 (1934). Tiselius, A., and Svensson, H., Tr. Faraday Xoc., 36, 16 (1940).

Downloaded from http://www.jbc.org/ by guest on April 2, 2019

1. The influence of a number of different buffers on the activity of p-amylase at 30” and an ionic strength of 0.05 was investigated over an approximate pH range of 3.8 to 6.2. 2. The pH optima for P-amylase activity in the presence of formate, acetate, propionate, butyrate, valerate, phenyl acetate, succinate, phthalate, citrate, and phosphate were determined. 3. Variation of the buffer anion was without significant influence on the relative activity of @-amylase at the pH optima, except for a slight inhibitory effect of phenyl acetate and phthalate. 4. The activity-pH curves approximately coincided on the alkaline side of the pH optima, whereas on the acid side a marked specific buffer influence was manifested. 5. A rapid and convenient method is described for the preparation from hard winter wheat of /3-amylase in the form of an active, water-soluble powder. 6. High concentrations of urea were found to inhibit the saccharogenic action of P-amylase on starch.

THE EFFECTS OF DIFFERENT BUFFERS ON THE ACTIVITY OF β -AMYLASE Gerald A. Ballou and J. Murray Luck J. Biol. Chem. 1941, 139:233-240.

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/139/1/233.citation.full.h tml#ref-list-1

Downloaded from http://www.jbc.org/ by guest on April 2, 2019

Access the most updated version of this article at http://www.jbc.org/content/139/1/233.citation