Studies on Methemoglobin Reductase: I. COMPARATIVE STUDIES OF DIAPHORASES FROM NORMAL AND METHEMOGLOBINEMIC ERYTHROCYTES

Abstract

Abstract Nonhemoglobin proteins of normal human red cells and methemoglobinemic red cells were separated from hemoglobin by carboxymethylcellulose column chromatography and NADH-dependent diaphorases were fractionated by the column chromatography on Sephadex G-75 and DEAE-Sephadex. Two NADH-dependent diaphorases, diaphorase A and diaphorase C, were isolated from the normal red cell extracts, diaphorase A forming 90% of the total activity. The Km of partially purified diaphorase A for 2,6-dichloroindophenol is 3.3 x 10-4 m. The diaphorase A fraction was less than 3% of normal in the methemoglobinemic red cell extracts. The methemoglobinemic enzyme is similar to the normal enzyme with respect to its Km for the dye, affinity with NADH, and the effects of phosphate on its activity, and, although its chromatographic behavior is normal, it is not identical with the normal enzyme, judging from its heat stability and electrophoretic properties. When assayed in 1.0 m sodium phosphate buffer, another NADH-dependent diaphorase, diaphorase B, is seen in both the normal and methemoglobinemic red cell extracts, which is found to be essentially NADPH-dependent diaphorase. It exhibits NADH-diaphorase activity in proportion to phosphate concentration and causes the phosphate activation of NADH-diaphorase activity in the methemoglobinemic hemolysate. When assayed in 1.0 m phosphate buffer, Km for the dye is 2.0 x 10-5 m and Km for NADH is 9.0 x 10-5 m regardless of NADH and the dye concentrations, respectively, which suggests a reaction mechanism whereby the affinity of one substrate to the enzyme is not influenced by the presence of another substrate.