Purification of human neutrophil NADPH oxidase cytochrome b-558 and association with Rap 1A

Abstract

The availability of sufficient quantities of highly purified phagocyte cytochrome b-558 has been necessary for many of the biochemical and immunological analyses of this important NADPH oxidase component, and it was only through the analysis of highly purified cytochrome b that the subunit composition was elucidated and the small subunit (p22-phox) was cloned and sequenced. In addition, the association of the small GTP-binding protein Rap1A with cytochrome b-558 was discovered through the analysis of purified cytochrome b. The procedures described here provide an easy, efficient, and highly reproducible method for the purification of cytochrome b as well as cytochrome b-Rap1A complexes. The ability to purify cytochrome b and cytochrome b-Rap1A complexes will also allow further analysis of the structure of this novel plasma membrane redox protein and the role of its association with low molecular weight GTP-binding proteins in the structure and regulation of the phagocyte NADPH oxidase.