CRYSTALLINE
ALCOHOL
DEHYDROGENASE YEAST*
FROM
BAKERS’
BY E. RACKER (From
the Department
of Microbiology, New College of Dentistry,
for publication,
New
University York)
October
College
of Medicin,e and
27, 1949)
Alcohol dehydrogenase, the enzyme which catalyzes the reversible oxidation of alcohol to acetaldehyde, was purified and crystallized from brewers’ yeast by Negelein and Wulff (1). The procedure described by these authors is laborious and time-consuming, and, particularly with American brewers’ yeast, the yield is small. It is the purpose of this paper to report the isolation and crystallization of alcohol dehydrogenase from bakers’ yeast by a simple procedure resulting in a yield of about 50 per cent of the enzyme activity present in the maceration juice. The crystalline enzyme can be obtained from the maceration juice within 1 working day. The procedure has been repeated in two other laboratories with similar success.l The enzyme is a useful tool for the study of dismutation reactions and for the determination of diphosphopyridine nucleotide (DPN), and has also been used as a model for kinetic studies of DPN-linked enzyme reactions. EXPERIMENTAL
Preparation of Crystalline Alcohol Dehydrogenase-Fleischmann’s bakers’ yeast was obtained in 1 pound cakes, crumbled, and dried between two sheets of paper in a thin layer for 4 to 5 days at room temperature. The dry yeast was finely ground in a ball mill at 0’ for 16 hours and stored in the cold room in a well stoppered container. 200 gm. of the dry yeast powder were extracted with 600 ml. of 0.066 M disodium phosphate for 2 hours at 37” with continuous stirring, followed by extraction at room temperature for an additional 3 hours. The yeast residue was then removed by centrifugation at 13,000 r.p.m. for 15 minutes.2 The supernatant solution was quickly brought to 55” and maintained at this temperature in a water bath for 15 minutes; after cooling, the mixture was centrifuged. At this stage the clear supernatant fluid can be stored at 0” overnight. * Aided by a grant from The National Foundation for Infantile Paralysis, Inc. i Personal communication from Dr. A. Kornberg, National Institute of Health, and Dr. S. Korkes, New York University College of Medicine.
2 The residue can be reextracted at least twice with considerable yields of alcohol dehydrogenase, which can be carried procedure used for the first extract.
through 313
to the crystalline
state by the same
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(Received
York
314
CRYSTALLINE
ALCOHOL
DEHYDROGENASE
TABLE
Purification
I
of Alcohol Dehydrogenase from Bakers’ Yeast Total
units
SpdfK
Yield
activity ---w&s per mg. )rotein
per cm:
lstextraot*................................... 55,000,OOOt 3,700 After heating. . . . . . . . . . . . . . . 62,000,OOO 100 55,000 90 Acetone ppt.................................. 56,000,OOO 1st crystalline preparation. . . . . . . . . 38,000,OOO 120,000 61 Recrystallized preparation. . . . . . . . . , . . . 26,000,OOO 158,000 42 --_____ -_____-* Prepared from 200 gm. of dried yeast. t The activity measurements of the first extract are usually low, probably due to side reactions occurring during the test.
the supernatant solution, the alcohol dehydrogenase enzyme started to crystallize a few minutes after the further addition of ammonium sulfate, which was added in small portions during the course of several hours until about 60 per cent of ammonium sulfate saturation was reached. The crystals were collected by centrifugation and resuspended in a small volume of distilled water, from which they started to recrystallize almost immediately on addition of small volumes of saturated ammonium sulfate solution. The recrystallized material was dialyzed against distilled water for 2 hours with rapid stirring and was then lyophilized. A typical protocol showing data on the specific activity of the various fractions and the yields obtained is presented in Table I. The dried enzyme has been stored for several weeks in a vacuum desicAccording to experiments described by cator with little loss in activity.
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To each 100 ml. of the yeast extract, 50 ml. of ice-cold acetone were slowly added, while the temperature of the mixture was maintained at about -2” in a dry ice-alcohol bath. The resulting precipitate was separated by centrifugation at 0” and discarded. To the supernatant solution, 55 ml. of cold acetone were added for each 100 ml. of the yeast extract, the temperature being kept at -2”. The mixture was centrifuged at 0” and the supernatant solution discarded. The precipitate was suspended in about 50 ml. of cold water and dialyzed for 3 hours against running tap water. The precipitate was removed by centrifugation. To the clear supernatant solution 36 gm. of solid ammonium sulfate were added per 100 ml. of solution. After standing at 0” for 30 minutes, the mixture was centrifuged at 15,000 r.p.m. for 20 minutes at 0”. The precipitate was dissolved in 20 ml. of distilled water and 4 gm. of ammonium sulfate were added. The precipitate was centrifuged off and discarded. From
E.
RACKER
315
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A. Kornberg,3 the enzyme can be stored in the frozen state and can be thawed repeatedly without marked loss of activity. For most of the studies reported in this paper, a solution of the lyophilized preparation was used. Spectrophotometric Measurement of Enzymatic Activity-The enzymatic activity of alcohol dehydrogenase was determined in a Beckman model DU quartz spectrophotometer with limiting amounts of enzyme and an excess of substrate. The enzyme preparation, diluted in 0.01 M phosphate buffer, pH 7.5, containing 0.1 per cent solution of gelatin or serum albumin to avoid surface denaturation, was added to a solution containing 0.01 M ethyl alcohol and 0.00005 M diphosphopyridine nucleotide. The final volume was 3 ml. and the final pH 8.8 with 0.01 M sodium pyrophosphate as buffer. The control cell contained all the solutions except the substrate. The changes in optical density at 340 rnp were recorded at intervals of 15 seconds. 1 unit is defined as a change in log lo/l of 0.001 per minute. The increment in density, log Jo/I, between the reading at 15 and 45 seconds after mixing of the solution, multiplied by 2, is taken as the enzyme activity per minute. Although the rate of DPN reduction falls off with time, the initial rates are proportional to the enzyme concentrations used. Determination of Diphosphopyridine Nucbotide (DPN)-In the presence of an excess of alcohol dehydrogenase (100 r) and a large excess of alcohol (1 mM), the reduction of DPN is very rapidly completed. The optical density obtained is directly proportional to the amount of DPN added and can be used to determine the DPN concentration in unknown solutions. From the equilibrium data presented later in this paper, it becomes apparent that, within the limits of experimental error, at an alkaline pH and in the presence of excess alcohol, DPN becomes fully reduced. This is confirmed by the fact that addition of semicarbazide, as used by previous investigators (1, 2) for the completion of the reaction, does not increase the amount of reduced DPN. Since DPN enters into this reaction stoichiometrically, at least 5 y of DPN must be present to obtain an accurate reading. DPN, even in amounts below 1 y, can be measured by means of alcohol dehydrogenase and yellow enzyme or diaphorase with semicarbazide as aldehyde fixative (1, 2). Enzymatic methods of DPN determination have the advantage of simplicity and specificity, in contrast to those methods depending on phosphorus determination or chemical reduction by hydrosulfite. Equilibrium Studies with Alcohol and Lactic Dehydrogenase-The use of alcohol dehydrogenase for the quantitative determination of diphosphopyridine nucleotide, in the absence of aldehyde fixatives such as semicarbazide, required a consideration of the equilibrium’of the reaction. * Personal communication.
316
CRYSTALLINE
ALCOHOL
DEHYDROQENASE
Alcohol
+ DPN+ C aldehyde + DPNH
+ H+
It is apparent from this formula that hydrogen ions take part in the reac-
tion and, therefore, that the pH at which the reaction is carried out will markedly affect the final equilibrium. High hydrogen ion concentration should favor the equilibrium of the reaction to the left, while low hydrogen ion concentration should shift the equilibrium to the right. To determine quantitatively the effect of H+ concentration on the equilibrium of the reaction, activity measurements were made at different pH levels. Measurements were carried out under the following conditions: 0.1 mg. of alcohol dehydrogenase, 30 PM of sodium pyrophosphate, 0.151 PM of diphosphopyridine nucleotide, and 16.2 PM of ethyl alcohol were added, and the pH adjusted by the addition of dilute NaOH or HCl. The final volume was 3 ml. The pH was measured with a glasselectrode at the end of each experiment. The equilibrium constants were determined by the following equations: lc = [acetaldehydel [DPNr,a.l [alcohol] [DPN,,.] k~
[acetaldehydel [DPNHI [a+] [alcohol] lDPN+I
(1) (2)
From the absorption at 340 rnp, the values for reduced DPN were calculated from the molecular absorption coefficient (6). For each molecule of DPN reduced, 1 molecule of alcohol is oxidized to acetaldehyde. The aldehyde concentration, therefore, is equal to the concentration of reduced DPN. The value for oxidized DPN was obtained by subtracting the amount reduced from the amount added. The alcohol concentration remained virtually constant, since less than 1 per cent could be oxidized by the amount of DPN present. When the Ic values were plotted on semilogarithmic paper against the pH at which the reaction took place, a straight line with a slope of 1 was obtained (Fig. 1). This can be readily understood from equation (2) as written above, in which the H ion concentration is included in the equilibrium formula. A shift of 1 pH unit (which is equivalent to a lo-fold
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Previous studies (3-5) have revealed a striking effect of pH on the equilibrium reaction catalyzed by enzymes requiring DPN. Negelein and Wulff (1) reported an equilibrium value, ([alcohol] [DPN,.]) / ([aldehyde] [DPN,,J) = 1350 at pH 7.9. No mea,surements at other pH values are recorded in their paper. The reaction catalyzed by alcohol dehydrogenase can be written as follows:
E.
317
RACKER
7.0
7.4
7.0
8.2 PH
8.8
9.0
9.4
FIG. 1. Effect of pH on the equilibrium constant k for alcohol dehydrogenase. The values for k are plotted on the ordinate.
and lactic
acid
DISCUSSION
Alcohol dehydrogenase with semicarbazide as an aldehyde fixative and in conjunction with yellow enzyme was first used for DPN determinations by Negelein and Wulff (1). From the equilibrium studies presented in the present paper it is apparent, however, that, at alkaline pH and with an excess of alcohol, DPN becomes fully reduced within the limits of experimental error without the use of substances which shift the equilib* A lactic acid dehydrogenase preparation rabbit muscle, was used for these studies.
of 40 per cent purity,
prepared
from
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change in H+ concentration), as expected, changes the value for the equilibrium constant by IO-fold. If the equilibrium is calculated with the inclusion of the H ion concentration, a constant kB is obtained at different pH values. For alcohol dehydrogenase, IcH = 1.15 X IO-“. The same relationship was found for lactic acid dehydrogenase4 at different pH values (see Fig. 1). The i& value = 4.4 X 10-12.
318
CRYSTALLINE
ALCOHOL
DEHYDROGENASE
SUMMARY
1. A simple method for the isolation of crystalline alcohol dehydrogenase from Fleischmann’s bakers’ yeast is described. 2. The enzyme is suitable for enzymatic determination of diphosphopyridine nucleotide. 3. Data on the equilibrium of the reaction catalyzed by the enzyme at diierent pH values are presented. 6 In that paper it was shown that with higher temperatures the equilibrium values for the lactate-pyruvate system increase. The values reported in this paper, which were determined at 25”, are slightly higher than those of Kubowitz and Ott at 22”.
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rium. Thus at pH 8.8, 0.1 PM of DPN is more than 98 per cent reduced in the presence of 1 PM of ethyl alcohol and an excess of alcohol dehydrogenase. The equilibrium data presented above show that coenzyme-linked reactions, such as those catalyzing the oxidation of alcohols to aldehydes or of lactate to pyruvate, can be measured directly in the spectrophotometer from either side of the reaction. Such measurements can also be made if the equilibrium appears unfavorable, provided the enzyme is not easily denatured in the pH range at which the test must be carried out. Both alcohol dehydrogenase and lactic acid dehydrogenase retained sufficient activity, even at a pH of 9.3. Since a great excess of enzyme can be used for these experiments, considerable losses in enzyme activity do not alter the final results of the equilibrium studies. Perhaps a few words of caution should be added in regard to the equilibria data. The use of an enzyme to obtain equilibria data presumes that the enzyme acts as a perfect catalyst and that the equilibrium reached is independent of the nature of the enzyme used. This was shown to be true for the succinate-enzyme-fumarate equilibrium measured by Borsook and Schott (7), but was not demonstrated in the above study. However, the amounts of alcohol dehydrogenase used are very small as compared to the substrate and the product of the reaction, and therefore are not likely to affect the equilibrium materially. The above reported results are in good agreement with the data previously obtained by Negelein and Wulff (1) with crystalline alcohol dehydrogenase and by Kubowitz and Ott (8) with crystalline lactic acid dehydrogenase.6 However, all these data show considerable variation from the results obtained with crude enzyme preparations (3, 4). For these reasons, it should be emphasized that the values obtained and the equation used may hold only for the particular conditions used in these experiments.
E.
RACKER
319
BIBLIOGRAPHY
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1. Negelein, E., and Wulff, H. J., Biochem. Z., 293,351 (1937). 2. Barron, E. S. G., in Enzymes and their r8le in wheat technology, New York, 193 (1946). 3. von Euler, H., Adler, E., and Hellstriim, H., 2. phusiol. Chem., 241,239 (1936). 4. von Euler, H., Adler, E., Gunther, E. G., and Hellstriim, H., 2. physiol. Chem., 245, 217 (1936). 5. Meyerhof, O., and Oesper, P., J. Bid. Chem., 170, 1 (1947). 6. Ohlmeyer, P., Biochem. Z., 297,66 (1938). 7. Borsook, H., and Schott, H. F., J. Biol. Chem., 92,535 (1931). 8. Kubowita, F., and Ott, P., Biochem. Z., 314,94 (1943).
CORRECTION On page 315, line 23, Vol. 184, No. 1, May, 1950, read 1 millimole
for 1 mM.
E. Racker J. Biol. Chem. 1950, 184:313-320.
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ARTICLE: CRYSTALLINE ALCOHOL DEHYDROGENASE FROM BAKERS' YEAST