PREPARATION AND CHARACTERIZATION OF ISOZYMES AND ISOFORMS OF HORSE LIVER ALCOHOL-DEHYDROGENASE

The procedure described allows the simultaneous large-scale preparatio n of the three main isozymes (EE, ES, SS) of alcohol dehydrogenase fro m horse liver (HLADH) and their subfractions using heat denaturation, ammonium sulfate precipitation, DEAF and CM ion-exchange chromatograph y as well as AMP-Sepharose affinity chromatography. Typical yields tha t can be obtained within three weeks are 1.5-2.5 g of EE-HLADH, 300-80 0 mg of ES-HLADH, 20-400 mg of SS-HLADH and 50-100 mg of EE-HLADH isof orms 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 bu t matrix-assisted laser desorption ionization mass spectrometry result ed in a molecular mass difference of + 130 to 165 relative molecular m ass. From a sugar determination and comparison of its pi with an artif icial glycosylation product of the EE-HLADH isozyme we concluded that the isoforms of HLADH are non-enzymatic glycosylation products which h ave been described to occur during protein aging.