[78] Liver alcohol dehydrogenase

[78] Liver alcohol dehydrogenase

[78] LIVER ALCOHOL DEHYDROGENASE 495 SUMMARY OF PURIFICATION PROCEDURE Volume, ml. Total protein, rag. Total units, thousands Specific activity...

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[78]

LIVER ALCOHOL DEHYDROGENASE

495

SUMMARY OF PURIFICATION PROCEDURE

Volume, ml.

Total protein, rag.

Total units, thousands

Specific activity, units/rag.

Yield, %

11.6 9.0 13.6 6.90 3.19 2.58 1.95

1.8 1.5 5.7 17.8 22.0 48 340

100 77 118 60 28 22 17

18.0 I0.5 8.8 10.4 9.5 8.2 3.84

2.9 1.7 3.7 20.0 31 56 410

100 59 49 58 53 45 21

Fraction A 1. 2. 3. 4. 5. 6. 7.

Cell extract 0.20-0.75 AS ~ Protamine supernatant 0.36-0.48 AS Heat-treated supernatant 0.40-0.44 AS Gel eluate

300 250 295 60 60 9 5

6300 5950 2380 387 143 54 57 Fraction B

1. 2. 3. 4. 5. 6. 7.

Cell extract 0.20-0.75 AS Protamine supernatant 0.60-1.0 AS Heat-treated supernatant 0.50-0.60 AS Gel eluate •

300 250 295 70 67 17 32

6300 5950 2380 520 308 144 9.4

Ammonium sulfate.

[78] Liver Alcohol Dehydrogenase A l c o h o l -t- D P N

~ ~ Acetaldehyde ~ DPNH ADH

~- H +

By ROGER K. BONNICHSEN a n d NORMAN G. BRINK Assay Method

Principle. R o u t i n e a c t i v i t y d e t e r m i n a t i o n a s d e s c r i b e d b y T h e o r e l l a n d B o n n i c h s e n 1 is b a s e d on s p e c t r o p h o t o m e t r i c m e a s u r e m e n t of t h e a m o u n t of D P N b e i n g r e d u c e d in 3 m i n u t e s a t p H 9.6 in t h e p r e s e n c e of excess of alcohol. Reagents 0.1 M g l y e i n e - s o d i u m h y d r o x i d e buffer, p H 9.6. D P N s t o c k s o l u t i o n c o n t a i n i n g a b o u t 10 mg. of p u r e D P N m i l l i l i t e r , o r i t s e q u i v a l e n t in less p u r e m a t e r i a l .

per

I H . Theorell and R. K. Bonnichsen, Acta Chem. •cand. 5, 1105 (1951); H. Theorell and B. Chance,/b/d. 5, 1127 (1951).

496

ENZYMES OF CARBOHYDRATE METABOLISM

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Ethanol, 96%. Enzyme. The enzyme should be diluted so that it contains 10 to 50 ~, of enzyme per milliliter.

Procedure. 3 ml. of the buffer, 0.1 ml. of the DPN solution, and 0.1 ml. of ethanol are mixed in a glass or plastic cell. 1 to 5 ~ of enzyme is added, and the optical density at 340 m~ is promptly read; t = 0. After 3 minutes the change of the optical density is recorded. Definition of Units. 1 "v of enzyme per milliliter gives in 1 cm. an increase in density of 0.045 in 3 minutes. The protein is measured at 280 mu; 1 rag. of enzyme per milliliter gives in a 1-cm. cell an optical density of 0.455. Application of Assay Method to Crude Tissue Preparations. The above method is used, but in this case a blank determination is also carried out. The change in density is measured under the same conditions but without addition of ethanol. This value is subtracted from the increase in density found in the presence of the substrate. Purification Procedure The following procedure is a modified form of the method described by Bonnichsen. 2 It has been carried out many times by different people and found to be reliable. The starting material is horse liver. Fresh materia[ is the best, but preparations of frozen liver have been successful. Step 1. The liver is ground it, a meat grinder. A Turmix should not be used. The liver is then extracted for 3 to 4 hours with double the amount of water. The extract can be left overnight at 4 ° . The extract is centrifuged or filtered conveniently through cheesecloth. Step 2. The red, turbid solution is heated to 52 ° and kept at this temperature for 15 minutes. It is important for further purification that this temperature be kept for fully 15 minutes. The solution is cooled to 20 to 25 ° and centrifuged or filtered. Step 3. This is a fractionation with ammonium sulfate at room temperature, the enzyme being collected between a saturation of 0.55 and 0.80. First, 349 g. of ammonium sulfate is added per liter of solution. After about 30 minutes the solution is centrifuged. It is important that the solution should be completely clear after the centrifugation. The precipitate is discarded, 208 g. of ammonium sulfate is added, and after 30 minutes the solution is centrifuged. The supernatant is discarded. The precipitate is dissolved in a small amount of phosphate buffer, pH 7, 0.01 M. The enzyme solution is then dialyzed against a phosphate buffer, pH 7, 0.01 M. The buffer should be changed several times, and I R. K. Bonnichsen, Acta Chem. Stand. 4, 715 (1950).

[78]

LIVER ALCOHOL DEHYDROGENASE

497

the dialysis continued until there is less than 2 % ammonium sulfate left in the enzyme solution. When the ammonium sulfate is added, small amounts of ammonia should be added to maintain the pH at about 6, as the enzyme is not stable below pH 5.40. At this point the only colored material the solution may contain is hemoglobin and very small amounts of catalase. Step 4. This step is intended to remove the hemoglobin. The method of Tsuchihashi a is used. To the dialyzed solution which contains 0.01 M phosphate, pH 7, is added 240 ml. of a alcohol-chloroform mixture (160ml. of alcohol and 80 ml. of chloroform) to each liter of solution, and the mixture is shaken vigorously for 10 minutes. The solution is centrifuged, and the denatured protein is discarded. The solution is immediately concentrated in vacuo. Most of the alcohol and the chloroform should be evaporated. It is convenient to concentrate to a small volume. The enzyme solution is dialyzed overnight against a phosphate buffer as above. After the dialysis the solution is centrifuged. The purity at this step should at least be 10%. It is possible at this point to get a rather pure enzyme by repeated ammonium sulfate fractionations. Step 5. The procedure involves a fractionation with ethanol at low temperature at the isoelectric point of the enzyme, pH 7. The ethanol used should be diluted to 90% v / v before use and chilled to - 10°. To the enzyme solution is added phosphate buffer, pH 7, to make the final concentration 0.02 M. It is then cooled to 0 °. The ethanol is added slowly, and the solution is cooled to - 5 to - 1 0 ° as soon as possible. The temperature should never be allowed to rise above 0 °. At 20 to 25 To of ethanol the enzyme begins to precipitate. The solution always contains some hemoglobin, and, as the enzyme is precipitated together with it, the hemoglobin color can be used as an indicator for the precipitation of the enzyme. The solution is allowed to stand for about an hour and is then centrifuged at - 1 0 °. The precipitate that contains the enzyme is suspended in ice-cold phosphate buffer, 0.02 M, pH 7. While the solution is being stirred, additional drops of buffer are added until all the hemoglobin is dissolved. The solution still remains cloudy. After some 30 to 60 minutes the enzyme begins to crystallize. It is left overnight in the cold room, and then the crystals are centrifuged at 0 °. The crystals are washed several times with cold phosphate buffer, 0.02 M, pH 7, containing about 6% ethanol. If the enzyme does not crystallize as described above, small amounts of ethanol can be added to the solution with continued stirring. If there is still difficulty in crystallizing the enzyme, it is probably because too much buffer has been used in dissolving the precipitate. In this case all the protein should be precipitated at - 1 0 ° with 30To ethanol, the '~M, Tsuchihashi, Biochem, Z, 140, 62 (1923),

498

ENZYMES OF CARBOHYDRATE METABOLISM

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precipitate this time taken up in a smaller a m o u n t of buffer, and the procedure described above repeated. The yield is about 800 mg. of crystalline enzyme from 5 kg. of liver.

Properties Specificity. In addition to ethanol, the enzyme reacts with higher aliphatic alcohols. Bliss 4 has shown t h a t it also oxidizes vitamin A.

I

310

320

I

!

3.30

340

.350

FIG. 1. Millimolar extinction of free and ADH-bound DPNH [H. Theorell and R. Bonnichsen, Aeta Chem. ,Scand. 5, 1105 (1951)]. Protein. The pure enzyme is colorless. The molecular weight is 73,000.1 The content of sulfur is 1.7%, of nitrogen 14.9%. The n u m b e r of free SH groups titrable with silver is seven per mole. 5 Inhibition. T h e enzyme depends on free SH groups for its activity and is inhibited b y SH-inhibiting reagents. C o n t r a r y to the yeast, A D H is not inhibited by 0.01 M iodoacetate. The enzyme is strongly inhibited b y hydroxylamine, s 4 A. F. Bliss, Arch. Biochem. and Biophys. 81, 197 (1951).

s R. K. Bonnichsen, in 4 Mosbacher Colloquiums, Springer Verlag, in press. 6N. O, Kaplan and M. M. Ciotti, J. Biol. Chem. 201, 785 (1953).

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LIVER ALCOHOL DEHYDROGENASE

499

pH Optimum. The Michaelis constant, K , , for alcohol varies with pH, reaching a minimum at pH 8, 5 X 10-~ M. With small substrate concentrations the pH optimum will be at the same pH. Mode of Action. One ADH molecule combines with 2 D P N H molecules at pH 7 and with 1 molecule at pH 10 to form a ADH-DPNH complex. 1 The spectrum of DPNH, as shown by Bonnichsen and Theorell$

6

// 4

a %.

%`

-1z a. {3

3:

cE ~ ¢q

t 0

I

!

I

I

O.2

0.4

0.6

0.8

1.0

FIO. 2. Spectrophotometric titration of ADH with increasing amounts of DPNH, pH 7 [H. Theorell and R. Bonnichsen, Acta Chem. Scand. 6, 1105 (1951)].

is changed by the complex formation, as seen in Fig. 1. This spectrum change can be used for an accurate determination of the enzyme. Figure 2 shows a titration of the ADH with DPNH. If the enzyme solution to be R. K. Bonnichsen and H. Theorell, see H. Theorell, 8e Conseil Chim. Inst. Intern. Solvay Bruxelles 395 (1950).

500

ENZYMES OF CARBOHYDRATE METABOLISM

[79]

tested is titrated with D P N H , the break of the curve as seen in the figure indicates when the solution is saturated with D P N H and when the a m o u n t of active enzyme present can be calculated.

["/91 Alcohol Dehydrogenase from Baker's Yeast R C H 2 0 H + D P N + ~ R C H O -}- D P N H + H +

By E. RACKER Assay Method 1

Principle. The m e t h o d is based on the absorption of D P N H at 340 m~. With ethanol in excess the rate of D P N reduction is proportional to enzyme concentration. Reagents 3 M ethanol. ~ 0.06 M sodium pyrophosphate, p H 8.5. 0.0015 M D P N 8 (enzymatically assayed). Enzyme. Dilute stock solution of enzyme in 0.1% bovine serum albumin in 0.01 M potassium phosphate, p H 7.5, to obtain about 200 to 1000 units of enzyme per milliliter.

Procedure. Place 2.2 ml. of distilled water, 0.5 ml. of pyrophosphate buffer, 0.1 ml. of e t h a n o l / a n d 0.1 ml. of D P N 3 into a quartz cell having a 1-cm. light path, and start the reaction b y addition of 0.1 ml. of enzyme. T h e control cell contains all reagents except the substrate. The first density reading is taken 15 seconds after addition of the substrate, and further readings are recorded at 15-second intervals. T h e increment in density between the 15- and 45-second readings times 2 is taken as enzyme activity per minute. Definition of [/nit and Specific Activity. One unit of enzyme is defined as t h a t a m o u n t which causes a change in optical density of 0.001 per minute under the above conditions. Specific activity is expressed as units 1E. Racker, J. Biol. Chem. 184, 313 (1950). 2 In the original paper 1the final alcohol concentration was erroneously given as 0.01 M instead of 0.1 M. 8 The amount of DPN used in this assay is not saturating but gives reproducible assay values.