Purification of Human Erythrocyte Pyruvate Kinase

Abstract

Abstract We describe a purification scheme for human erythrocyte pyruvate kinase. Following two ammonium sulfate fractionations, batchwise absorption on CM-cellulose, chromatography on CM-cellulose with fructose 1,6-diphosphate elution, and chromatography on DEAE-cellulose, a 30,000-fold purification was achieved with a yield of 8% from the original hemolysate. Antiserum against partially purified pyruvate kinase was prepared in rabbits and used to follow the subsequent purification of the enzyme. Catalase and immunoglobulin G were identified by immunodiffusion as components of the impure preparation. Isoelectric focusing of partially purified pyruvate kinase gave a single activity peak with an isoelectric point of 7.1 at 4°. The final product was homogeneous as judged by polyacrylamide disc gel electrophoresis and immunodiffusion. No free NH2-terminal amino acids could be detected. Sedimentation velocity analysis gave a major peak and a faster migrating, minor peak corresponding to 5% of the total. We suggest that the minor peak is due to aggregation of the major component. From sedimentation equilibrium analysis a molecular weight of 225,400 was obtained for the purified pyruvate kinase. The amino acid composition is presented.