Preparation and characterization of isozymes and isoforms of horse liver alcohol dehydrogenase

The procedure described allows the simultaneous large-scale preparation of the three main isozymes (EE, ES, SS) of alcohol dehydrogenase from horse liver (HLADH) and their subfractions using heat denaturation, ammonium sulfate precipitation, DEAE and CM ion-exchange chromatography as well as AMP-Sepharose affinity chromatography. Typical yields that can be obtained within three weeks are 1.5-2.5 g of EE-HLADH, 300-800 mg of ES-HLADH, 20-400 mg of SS-HLADH and 50-100 mg of EE-HLADH isoforms from 5 kg of horse liver. The EE-HLADH isoform prepared has a pI of 7.8, which is 0.3 pH units lower as compared to the main fraction; the zinc content and number of free sulfhydryl groups are unchanged but matrix-assisted laser desorption ionization mass spectrometry resulted in a molecular mass difference of + 130 to 165 relative molecular mass. From a sugar determination and comparison of its pI with an artificial glycosylation product of the EE-HLADH isozyme we concluded that the isoforms of HLADH are non-enzymatic glycosylation products which have been described to occur during protein aging.