Immunological studies on the respiratory burst oxidase of pig blood neutrophils

Recently, a flavin enzyme (pI 5.0), that is probably responsible for superoxide (O2-)-generated oxidase activity, was separated by isoelectric focusing-polyacrylamide gel electrophoresis (IEF-PAGE) from neutrophil membranes in our laboratory [(1987) J. Biol. Chem. 262, 12316-12322]. In the present work, we performed immunological studies on this enzyme derived from pig blood neutrophils. The enzyme extract obtained on IEF-PAGE was injected into guinea pigs to raise antibodies. IgG antibody against the pI 5.0 protein inhibited maximally 54% of the O2- -generating activity of the membrane-solubilized oxidase, whereas the normal serum IgG was not inhibitory at all. Our results further confirmed that the enzyme (PI 5.0) is one of the component(s) of the O2- -generating system. The enzyme gave rise to a band corresponding to a major protein of 72 +/- 4 kDa on both non-denaturing and SDS-PAGE. Immunoblotting after SDS-PAGE demonstrated labelling of peptides of 70-72, 28-32 and 16-18 kDa.