84] Phosphoglucose isomerase of human erythrocytes and cardiac tissue

Abstract

This chapter describes the assay method, purification procedure, and properties of phosphoglucose isomerase of human erythroeytes and cardiac tissue. Enzyme activity can be measured in either the forward or the reverse direction by using the appropriate coupling enzymes and monitoring the oxidation or reduction of NADH or NADP spectrophotometrically. The isomerase activity is more conveniently assayed in the reverse direction by coupling with glucose-6-phosphate dehydrogenase and following the rate of reduction of NADP. The forward reaction can be measured by coupling with phosphofructokinase, aldolase, triosephosphate isomerase, and α -glycerophosphate dehydrogenase and monitoring the rate of oxidation of NADH. The purity of both substrates (fructose 6-phosphate and glucose 6-phosphate as substrates) and all coupling enzymes is adequate to prevent baseline drift or interference during the assay. In either the forward or reverse assays, the substrates are used as their sodium salts, and the concentrations of substrates are determined enzymically. The method is discussed that has been developed for the rapid isolation of human phosphoglucose isomerase by substrate elution from phosphocellulose. All steps carried out at 0–4° and 0.1% v/v 2-mercaptoethanol is included in all buffer solutions. The physical, chemical, and catalytic properties of the enzyme isolated from erythrocytes are identical with the enzyme isolated from muscle tissue. Although erythrocytes are often the only available source of material for the isolation, muscle tissue is preferred, due to its higher concentration of the enzyme.