Purification of Pyruvate Dehydrogenase by Affinity Chromatography
The pyruvate dehydrogenase complex of Ezchen.Lchia cotL consists of three enzymes: pyruvate decarboxylase (El) (EC 126.96.36.199), lipoate acetyltransferase (E2) (EC 2.3. 1.12), and lipoamide dehydrogenase (E3) (EC 188.8.131.52). Various purification procedures exist; but the one commonly used (1) involves protamine sulfate fractionation and isoelectric precipitations at pH 5.7 to remove the a-oxoglutarate dehydrogenase complex and at pH 5.0 to precipitate the pyruvate dehydrogenase complex. A further improvement was obtained by introducing several intermediate isoelectric precipitation steps and calcium phosphate gel-cellulose chromatography (2). Another method (3) is more rapid and uses chromatography on Biogel A-50-M followed by calcium phosphate gel-cellulose chromatography. We recently developed a method based on Biogel chromatography followed by affinity chromatography on thiamin pyrophosphate, but due to instability of the matrix this method is of limited value. In the study being reporteda rapid and mild purification procedure is described using ethanolamine as a ligand.
KeywordsMagnesium EDTA Electrophoresis Fractionation Pyruvate
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