Purification and Characterization of Sorbitol Dehydrogenase from Bovine Brain

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Abstract
Sorbitol dehydrogenase (EC 1.1.1.14) was isolated from bovine brain and purified 3,000‐fold to apparent homogeneity, as judged by polyacrylamide gel electrophoresis. The purified enzyme had a specific activity of 36 units/mg of protein; a molecular weight of 39,000 for each of the four identical subunits and 155,000 for the intact enzyme were determined by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and gel exclusion chromatography, respectively. The presence of one Zn2+ per subunit was confirmed by atom absorption spectroscopy; inactivation of the enzyme by metal‐chelating agents points to the essential role that Zn2+ plays in the catalytically competent enzyme. The enzyme is also inactivated by thiol‐blocking reagents; with respect to inactivation by sodium pyrophosphate, sorbitol dehydrogenase is different from closely related alcohol dehydrogenase.