The Assay and Specific Activity of Crystalline Alcohol Dehydrogenase of Horse Liver.

Abstract

A straight-line relation was demonstrated when varying amts. of cryst. alc. dehydrogenase of horse liver (Colowick and Kaplan, Methods in Enzymology, 1955, 835 pp. (C.A. 49, 11768i)) (I) were plotted against 1/t0.2, in which t0.2 represents the time required for the increase of 0.200 in E340, i.e., the reduction of a definite fraction of diphosphopyridine nucleotide (DPN). The reaction mixt., conducted in a cuvet 1-cm. deep, consisted of 1.85 ml. of 0.1M glycine-NaOH buffer, pH 10.0, 1.0 ml. of DPN soln. (0.83 mg. of cozymase 90/ml.), and 0.15 ml. of EtOH (1 ml. of 96% EtOH/ml. H2O), to which was added 0-15 γ of the I prepn. recrystd. 5 times with 7-10% EtOH to const. activity/Ε280 ratio. In the activity test of Theorell and Bonnichsen (C.A. 45, 9093c), 1 γ of I prepn./ml. gave an increase of Ε340 of 0.058 in 3 min., a value significantly higher than those previously reported. A 2nd component, comprising about 10% of the I prepn., was observed after prolonged electrophoresis. [on SciFinder(R)]