STOICHIOMETRY OF P22-PHOX AND GP91-PHOX IN PHAGOCYTE CYTOCHROME B(558)

The phagocyte NADPH oxidase complex is an unusual electron transfer sy stem. Its principal component, cytochrome b(558), is a heme-containing integral membrane protein consisting of two subunits, gp91-phox and p 22-phox. We used a novel method to measure precisely the gp91-phox:p22 -phox stoichiometry. Cytochrome b(558) was isolated in high purity fro m human neutrophil membrane preparations using a novel affinity purifi cation method. We performed direct peptide sequencing of purified cyto chrome b(558) and detected two amino acid sequences which matched pred icted sequences for gp91-phox and p22-phox. We quantitated amounts of both amino acids released from p22-phox and gp91-phox in each sequenci ng cycle. Averaging over 25 . cycles, the mean p22-phox:gp91-phox rati o of released amino acids was 0.93 +/- 0.01. To correct for recovery d ifferences between individual amino acids, we measured individual p22- phox:gp91-phox ratios for the eight different amino acids common to bo th p22-phox and gp91-phox in the first 25 positions. The mean of indiv idual p22-phox:gp91-phox ratios for the eight common amino acids was 0 .96 +/- 0.05. The p22-phox:gp91-phox ratios for each of the eight comm on amino acids varied from 0.81 to 1.20. Taken together, measured rati os for total and individual amino acids are consistent with a predicte d ratio of 1.0 for 1:1 p22-phox:gp91-phox stoichiometry in cytochrome b(558).