Catalog Number: 100161, 151430, 159858 Alcohol Dehydrogenase

Molecular Weight: 141,000 comprised of four subunits of 35,000 each.8

CAS # : 9031-72-5

Synonyms: ADH; Alcohol : NAD+ oxidoreductase

Source: Yeast

E.C. 1.1.1.1

Description: Alcohol dehydrogenase catalyzes the reaction:

The common reaction in yeast cells is reduction of acetaldehyde to ethanol. In vitro the enzyme is generally assayed and used in a more alkaline pH region, a condition which favors a shift of equilibrium towards the oxidation of ethanol.

Composition: A metalloenzyme containing four tightly bound zinc atoms per molecule.18,19 Per subunit, there are two distinct active site sulfhydryl groups which can be distinguished on the basis of differential reactivity with iodoacetate and butyl isocyanate.17 A histidine residue is considered to have an essential role.9

Optimum pH: For the oxidation of ethanol, pH 8.6-9.0 (the enzyme becomes increasingly unstable at higher pH). For the reduction of acetaldehyde a pH nearer to 7.0 is considered optimum. This reaction is kinetically complex with pH being only one factor determining optimum conditions.

1% Extinction coefficient: E 260= 12.6.

Isoelectric point: 5.4.16

Solubility: Dissolves readily at 5 mg/ml in 0.01 M sodium phosphate pH 7.5 to give a clear colorless solution.

Activators: Sulfyhydryl activating reagents, mercaptoethanol, dithiothreitol, cysteine, etc., and heavy metal chelating reagents.

Specificity: Yeast ADH which has a more narrow specificity than that of liver enzyme, accepts ethanol, is somewhat active on the straight chain primary alcohols, and acts to a very limited extent on certain secondary and branched chain alcohols.10 NADP does not serve as coenzyme.

Inhibitors: Heavy metals and -SH reagents. See Sund and Theorell16 for a more comprehensive list; also Anderson and Reynolds2, Anderson et al.3, Atkinson et al.5, Fiddick and Heath11, Rashed and Rabin15.

Stabilizers: Dilute solution of the enzyme may be stabilized by serum albumin, gelatin, and/or glutathione or cysteine. At pH values below 6.0 and above 8.5 the enzyme is increasingly unstable. More concentrated solutions of the enzyme in high purity water, near neutrality, are stable several days at 5oC.

Assay

Method: The reaction velocity is determined by the method of Vallee and Hoch18,19 in which the rate of absorbance at 340 nm resulting from reduction of NAD is measured. One unit reduces one micromole of NAD per minute at 25oC under the specified conditions.

Reagents 0.1 M Sodium pyrophosphate buffer, pH 9.2 2 M Ethanol. Dilute 12.12 ml of 95% ethanol to 100 ml with reagent grade water. 0.025 M NAD. Note: NAD may vary in salt form and degree of hydration. Care should be exercised to use an analytical grade and the correct molecular weight. 0.1 M Phosphate buffer, pH 7.5 0.1% Albumin (BSA) in 0.01 M phosphate buffer, pH 7.5

Enzyme

Dissolve lyophilized enzyme at one mg/ml in 0.1 M phosphate buffer, pH 7.5. Immediately prior to use, dilute all enzymes to a concentration of 0.05-0.25 units/ml in 0.1% albumin.

For yeast enzyme: mg/ml = A260 x 0.796

For protein determinations, dissolve enzyme in 0.1 M phosphate buffer, pH 7.5.

Procedure

Set spectrophotometer to 340 nm and 25oC.

Pipette into each cuvette:

0.1 M Pyrophosphate buffer 1.5 ml 2.0 M Ethanol 0.5 ml 0.025 M NAD 1.0 ml

Incubate in spectrophotometer for 3-4 minutes at 25oC to achieve temperature equilibrium and establish blank rate, if any. At zero time, add 0.1 ml of appropriately diluted enzyme to the cuvette and record the A340 for 3-4 minutes. Calculate the A340/minute from the initial linear portion of the curve.

Calculation

Units/mg protein = (DA340/min) / (6.22 x mg protein/ml reaction mixture)

Availability:

Catalog Number Description Size 100161 Alcohol Dehydrogenase, 2X crystallized, 7.5 KU lyophilized. Activity: approximately 300 15 KU units/mg 30 KU 75 KU 150 KU 500 KU 151430 Alcohol Dehydrogenase, lyophilized 25 mg powder. Activity: not less than 300 units/mg 250 mg protein 500 mg 159858 Alcohol Dehydrogenase, 2X crystallized, 100 mg suspension in 2.4 M ammonium sulfate 500 mg containing 3% pyrophosphate and 1% glycine

References: 1. Ainslie, G., and Cleland, W., "Isotope Exchange Studies on Liver Alcohol Dehydrogenase with Cyclohexamol and Cyclohexanone as Reductants," J. Biol. Chem., v. 247, 946 (1972). 2. Anderson, B., and Reynolds, M., "Multiple Inhibition of Yeast Alcohol Dehydrogenase by Heterocyclic Nitrogen Bases," Arch. Biochem. Biophys., v. 114, 299 (1966). 3. Anderson, B., Reynolds, M., and Anderson, C., "Nitrogen Base Inhibition of Yeast Alcohol Dehydrogenase," Biochim. Biophys. Acta, v. 113, 235 (1966). 4. Andersson, P., Kvassman, J., Oldén, B., and Pettersson, G., "Electrostatic Field Effects of Coenzymes on Ligand Binding to Catalytic Zinc in Liver Alcohol Dehydrogenase," Eur. J. Biochem., v. 138, 603 (1984). 5. Atkinson, M., Eckermann, G., and Lilley, R., "Inhibition of Alcohol Dehydrogenase from Yeast by Pyridine," Biochem. J., v. 104, 872 (1967). 6. Baillie, A., Calman, K., and Hart, D., "Histochemical Distribution of Alcohol Dehydrogenases in Endocrine Tissue," Nature, v. 210, 1277 (1966). 7. Brändén, C., Jörnvall, H., Eklund, H., and Furugren, B., "Alcohol Dehydrogenases" in The Enzymes, XI, 3rd ed., Part A, (Boyer, P., ed.), Academic Press, NY, 103 (1975). 8. Büchner, M., and Sund, H., "Yeast Alcohol Dehydrogenase: -SH Groups, Di-sulfide Groups, Quaternary Structure, and Reactivation by Reductive Cleavage of Disulfide Groups," Eur. J. Biochem., v. 11, 73 (1969). 9. Dickenson, C., and Dickinson, F., "The Role of an Essential Histidine Residue of Yeast Alcohol Dehydrogenase," Eur. J. Biochem., v. 52, 595 (1975). 10. Dickinson, F., and Dalziel, K., "Substrate Specificity and Stereospecificity of Alcohol Dehydrogenases," Nature, v. 214, 31 (1967). 11. Fiddick, R., and Heath, H., "Inhibition of Alcohol Dehydrogenase by Chloroquine," Nature, v. 213, 628 (1967). 12. Jörnvall, H., "Partial Similarities Between Yeast and Liver Alcohol Dehydrogenases," Proc. Natl. Acad. Sci. USA, v. 70, 2295 (1973). 13. Myers, J., and Jakoby, B., "Glycerol as an Agent Eliciting Small Conformational Changes in Alcohol Dehydrogenase," J. Biol. Chem., v. 250, 3785 (1975). 14. Pereira, E., Aracava, Y., Aronstam, R., Barreiro, E., and Albuquerque, E., "Pyrazole, an Alcohol Dehydrogenase Inhibitor, Has Dual Effects on Normal-Methyl-D-Aspartate Receptors of Hippocampal Pyramidal Cells - Agonist and Noncompetitive Antagonist," J. Pharm. Exp. Ther., v. 261, 331 (1992). 15. Rashed, N., and Rabin, B., "Inhibition of Yeast Alcohol Dehydrogenase by Alkylating Agents," Eur. J. Biochem., v. 5, 147 (1968). 16. Sund, H., and Theorell, H., "Alcohol Dehydrogenases" in The Enzymes, VII, 2nd ed., (Boyer, P., Lardy, H., and Myrback, K., eds.), Academic Press, NY, p. 25 (1963). 17. Twu, J., Chin, C., and Wold, F., "Studies on the Active-Site Sulfhydryl Groups of Yeast Alcohol Dehydrogenase," Biochem., v. 12, 2856 (1973). 18. Vallee, B., and Hoch, F., J.A.C.S., v. 77, 821 (1955). 19. Vallee, B., and Hoch, F., "Zinc: A Component of Yeast Alcohol Dehydrogenase," Proc. Natl. Acad. Sci. USA, v. 41, 327 (1955).