Properties of cytochrome b5 and methemoglobin reduction in human erythrocytes.

Abstract

Cytochrome b5 of human erythrocytes showed a midpoint redox potential of - 2 mV, an isoelectric point of 4.3, and a Michaelis constant of 7μ for erythrocyte cytochrome b5 reductase These values are close to those of rabbit liver cytochrome b5, though cytochrome b5 of erythrocytes is in the soluble fraction and its relative molecular mass is smaller than that of liver cytochrome b5 The concentration of cytochrome b5 in normal human erythrocytes was determined by an improved method and a value of 0.22 ± 0.02 μM was obtained. The optimum pH of the cytochrome b5 reduction by the reductase from human erythrocytes was 5.5, and about half of the maximum activity was observed at neutral pH. As electron donors, α-NADH and NADPH were 56% and 1 % as effective as β-NADH respectively. A Michaelis constant of 22 nM for β-NADH was obtained by fluorometric assay method. The rate of cytochrome b5 reduction in normal human erythrocytes was calculated to be 1.4 mM/h by the use of the substrate concentrations in vivo. The rate constant of the nonenzymatic methemoglobin reduction by reduced cytochrome b5 was determined to be 6.2 × 103 M-1 s-1. These values account for the methemoglobin reduction rate in human erythrocytes on the hypothesis proposed previously (Hultquist, Sugita), namely methemoglobin is reduced nonenzymatically by cytochrome b5 which has been reduced by NADH-cytochrome b5 reductase.