Stoichiometry of the subunits of flavocytochrome b558 of the NADPH oxidase of phagocytes

Abstract

Flavocytochrome b558, the membrane-spanning component of the NADPH oxidase system of phagocytic cells, is composed of two subunits, p22phox and gp91phox (where phox stands for phagocyte oxidase). The stoichiometry of the subunits has been determined for purified flavocytochrome b558 by: (1) densitometry of Coomassie Blue-stained proteins separated by SDS/PAGE, (2) aromatic absorbance at 280 nm by the subunits after separation by gel filtration under denaturing conditions, (3) cross-linking studies with bis[sulphosuccinimidyl]suberate, where the molecular mass of the cross-linked complex was determined by Western blotting, and (4) radiolabelling of pure flavocytochrome b558 on lysine residues with 125I-labelled Bolton–Hunter reagent (N-succinimidyl-3-(4-hydroxy-5-[125I]iodophenyl)propionate) followed by SDS/PAGE and determination of the radioactivity on each subunit. The ratio of p22phox to gp91phox in the purified flavocytochrome b558 was related back to that in the neutrophil membrane by quantitative Western and dot-blotting to ensure that the stoichiometry was maintained during purification. These measurements showed that the two subunits were present in neutrophil membranes in a molar ratio of 1:1.