New Simple Method for Purification of Class I Alcohol Dehydrogenase

AIMS: The purpose of this study was to develop a new simple method for purification of rat class I alcohol dehydrogenase (ADH, EC 1.1.1.1). METHODS AND RESULTS: Immobilized p-hydroxyacetophenone was used as a ligand for affinity chromatography for the initial purification step after ammonium sulfate precipitation of the cytosolic fraction of rat liver. Then the eluant was separated by using ion-exchange chromatography, and homogenous class I ADH, as judged by the results of SDS-PAGE and confirmed by the results of the amino-acid sequence of peptides degraded from a 39 kDa protein, was obtained with a high yield (57%). The purified ADH showed kinetic constants of 1.3 mmol/l for Km and 62.4 per min for Kcat with ethanol as a substrate. ADH was also successfully purified from yeast by a similar method using p-hydroxyacetophenone affinity chromatography. CONCLUSIONS: This simple method involving only two chromatographic procedures may be very useful for purification of ADH.