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Alcohol & Alcoholism Vol. 39, No. 3, pp. 178-182, 2004
Alcohol & Alcoholism Vol. 39, No. 3 © Medical Council on Alcohol 2004; all rights reserved

NEW SIMPLE METHOD FOR PURIFICATION OF CLASS I ALCOHOL DEHYDROGENASE

M. Negoro and I. Wakabayashi*

Department of Hygiene and Preventive Medicine, Yamagata University School of Medicine, Yamagata, Japan

* Author to whom correspondence should be addressed at: Department of Hygiene and Preventive Medicine, Yamagata University School of Medicine, Iida-Nishi 2-2-2, Yamagata 990-9585, Japan. Tel.: +81 23 628 5252; Fax: +81 23 628 5255; E-mail: wakabaya{at}med.id.yamagata-u.ac.jp

(Received 1 September 2003; first review notified 25 November 2003; in revised form 26 December 2003; accepted 29 December 2003)

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.


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