Evaluation of the Cytochrome b5/Cytochrome b5Reductase Pathway

NADH cytochrome b5reductase (b5R; EC 1.6.2.2; Diaphorase I; NADH: ferricytochrome b5oxidoreductase) is an FAD‐containing protein, which, along with the hemoprotein cytochrome b5(cyt b5), mediates electron transfer from NADH to fatty acid desaturases, P450 oxidases, methemoglobin, and ascorbyl free radical. In addition, b5R and cyt b5can directly catalyze the reduction of hydroxylamine and amidoxime metabolites. This unit provides protocols for measuring the activity and mRNA expression of the cytochrome b5/cytochrome b5reductase pathway, and for obtaining heterologous expression and purification of the soluble forms of each protein.