Schematic representation of the isolation of active cytochrome b558 from heparin agarose matrix procedure. Cytochrome b558 was extracted with 2% (w/v) octyl-glucoside from the membrane of stimulated human neutrophils as described in Materials and Methods. Proteins of the soluble extract were loaded onto a mixture of CM, DEAE, and n-amino-octyl Sepharose combined to heparin agarose. Cytochrome b558 bound to the heparin affinity matrix. The matrix was extensively washed with either rS100A9-A8 chimera or with cytosol of stimulated EBV-B lymphocytes. The cytochrome b558 containing fractions eluted from heparin agarose were pooled and filtrated on S-300 Sephacryl. Purified cytochrome b558 recovered from Sephacryl displayed a constitutive NADPH oxidase activity. (TIF)