Purification and properties of soluble NADH-cytochrome b5 reductase of rabbit erythrocytes.

Soluble NADH-cytochrome b5 reductase was purified from rabbit erythrocytes to homogeneity by simple procedures developed in this study including fractionation with ammonium sulfate, gel filtration on a Sephadex G-75 column, and affinity chromatography on a 5'-AMP-Sepharose 4B column. The enzyme was purified about 12,000-fold from hemolysate in terms of NADH-cytochrome b5 reductase activity with a high yield of 40%. The purified enzyme has absorption maxima at 273, 390, and 462 nm, and shoulders at 370, 435, and 488 nm. The ratio of the absorbance at 273 nm to that at 462 nm of the purified enzyme was 5.6-5.8. The prosthetic group of the enzyme was found to be FAD, and the flavin content in the enzyme was 1 mol/mol of the enzyme. The molecular weight of the purified enzyme was estimated to be 33,000 and 32,000 by gel filtration on a Sephadex G-75 column, and by electrophoresis on polyacrylamide gel in the presence of sodium dodecyl sulfate, respectively. The NADH-cytochrome b5 reductase activity decreased strikingly as the buffer or salt concentration in the assay mixture was increased, and the optimal pH for the reduction of cytochrome b5 with NADH was determined to be 6.6 in Tris-maleate buffer of constant ionic strength. The maximum velocity of NADH-cytochrome b5 reductase activity of the purified enzyme was very high, 1,280 mumol/min/mg of protein in 10 mM phosphate buffer (pH 6.6). The Michaelis constants for NADH and cytochrome b5 were determined to be 2.5 and 4 microM, respectively. The reduction of cytochrome b5 with NADH by the enzyme was suggested to follow the ordered-type reaction mechanism based on the modes of product inhibition. From these results, and also from the estimated enzyme content in the erythrocytes (16-20 mg protein per liter of packed erythrocytes), the possible contribution of the enzyme to functions other than methemoglobin reduction in rabbit erythrocytes is discussed.