Abstract
A novel procedure was developed for rapid separation of the three component enzymes of pig heart 2-oxoglutarate dehydrogenase complex by high performance liquid chromatography on a gel filtration column. The complex was dissociated and separated into two fractions of the first dihydrolipoamide succinyltransferase and a second yellow fraction within 1 h by chromatography on a preparative TSK-GEL G4000SW column equilibrated with 0.05 M potassium phosphate buffer (pH 7.0) containing 0.7 M guanidine hydrochloride, 0.05% Triton X-100 and 2 mM dithiothreitol at 10 degrees C. The dihydrolipoamide succinyltransferase fraction was further purified by incubation with 0.5% sodium deoxycholate and subsequent ammonium sulfate fractionation. The other two component enzymes, 2-oxoglutarate dehydrogenase and lipoamide dehydrogenase were separated from the second yellow fraction by chromatography on a calcium phosphate gel-cellulose column. The TSK-GEL column permitted very rapid dissociation and separation of the three component enzymes accompanied by good preservation of their activities and high overall yields.
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