A convenient large scale preparation of the EE isozyme of horse liver alcohol dehydrogenase

Abstract

A modified method suitable for the preparation of large amounts of very pure EE isozyme of horse liver alcohol dehydrogenase is described. The enzyme was purified from fresh horse livers by ammonium sulfate precipitation, ion-exchange, and gel-filtration chromatography. The method has been used for large as well as small-scale purifications: a 5-g yield of pure EE isozyme is possible from 14 kg of fresh liver at 140% of the specific activity previously reported. Isoelectric focusing revealed a single band stained with Coomassie blue, and there was essentially no activity using an assay for steroid-active subunits. If stored under sterile conditions the purified enzyme is stable for 6 months and should be particularly suitable for investigations of potential site heterogeneity of the enzyme, an area of current controversy.