CONSIDERATIONS IN EVALUATING ZINC CONTENT OF HORSE LIVER ALCOHOL DEHYDROGENASE PREPARATIONS

Since horse liver alcohol dehydrogenase was first reported to contain 2 g-atoms of Zn/mol wt 73 × 103, the methods for purification of the enzyme and its molecular weight have been revised repeatedly. The specific activity of the product has increased substantially, resulting in uncertainties concerning the molar stoichiometry of metal and protein. The present paper reviews the results of zinc analyses of horse liver alcohol dehydrogenase preparations in this laboratory since the inception of work on this enzyme. The observed variations in zinc content are outside the limits of confidence of the methods employed for metal analysis. However, improvements in horse liver alcohol dehydrogenase isolation and characterization, including molecular weight determinations and recognition of the subunit structure and isoenzymes of horse liver alcohol dehydrogenase, all appear to bear on the variations in molar stoichiometry observed over the years. The zinc contents of recently obtained and uniformly treated samples of horse liver alcohol dehydrogenase vary from 3.1 to 4.3 g-atoms of Zn per mole of horse liver alcohol dehydrogenase, based on a molecular weight of 80 × 103. A variety of conditions alter both the zinc content and the catalytic activity of the enzyme. Only 2 g-atoms of Zn/mole of horse liver alcohol dehydrogenase appear to be directly related to enzymatic activity, as shown by selective removal of zinc from horse liver alcohol dehydrogenase, confirming the existence of two active-site zinc atoms. © 1969, American Chemical Society. All rights reserved.