Purification of Isoenzyme and Coenzymes for Kinetic Study of Horse Liver Alcohol Dehydrogenase

Abstract

The isoenzymic forms of the alcohol dehydrogenase (EC 1.1.1.1) of horse liver have been separated by chromatography on carboxymethylcellulose column and identified in terms of their electrophoretic mobilities in the starch gel. Purified isoenzyme fraction C₁, which was highest in both total and specific activities, was stable toward rechromatography on carboxymethylcellulose, electrophoresis in starch gel, as well as storage in solution. It therefore satisfied the requirements for use in kinetic studies on the reaction mechanism of the enzyme. Toward the same end, procedures are also described for preparing purified NAD⁺ and NADH in solution forms which were stable in storage for several days.