Characterization of the purified NADH-cytochrome b5 reductase of human erythrocytes as a FAD-containing enzyme.

NADH-cytochrome b5 reductase of normal human erythrocytes was purified by procedures including affinity chromatography on Blue-Sepharose to an electrophoretically homogeneous protein. The purified enzyme was judged to be a typical flavoprotein based on its absorption spectrum (absorption maxima, 272, 390, and 462 nm; shoulders, 373 and 488 nm) and flavin content (1 mol of FAD/mol of enzyme). The minimum molecular weight calculated from the flavin content was 32,300. The purified enzyme showed a distinct negative circular dichroic spectrum at 280 nm and also at 460 to 480 nm. With the best preparations, the molar ellipticities at 280 and 460 nm were well correlated with the enzyme activity and flavin content in the enzyme. A partial loss of flavin from the enzyme led to a concomitant loss of enzyme activity and decrease in the molar ellipticities at 460 and 280 nm. Flavin analogues such as acrinol and proflavine (0.1 mM) strongly inhibited the enzyme activity, and atebrin (0.1 mM) also showed partial inhibition. Complete inhibition was observed with 1 mM of any these reagents. These results apparently indicate that FAD in the enzyme functions as a prosthetic group, and that circular dichroic spectroscopy is a good measure of the bound form of flavin in the enzyme.