Immunoaffinity Purification of Human Phagocyte Flavocytochrome b and Analysis of Conformational Dynamics

Abstract

The heterodimeric integral membrane protein flavocytochrome b (Cyt b) is the catalytic core of the phagocyte NADPH oxidase, an enzyme complex that initiates a cascade of reactive oxygen species critical for the elimination of infectious agents. Many fundamental questions remain concerning the structure and catalytic mechanism of Cyt b, largely because of the inability to isolate this protein in quantities required for both biochemical analysis and meaningful attempts at high-resolution structure determination. In order to facilitate the direct analysis of Cyt b, the following method describes a rapid and efficient procedure for the immunoaffinity purification of Cyt b (under nondenaturing conditions) from neutrophil membrane fractions. The protocol presented here contains a number of steps that have been optimized and improved since the original description of this Cyt b isolation method. In order to address questions concerning the mechanism of superoxide generation by the NADPH oxidase complex, methods are additionally presented for analysis of conformational dynamics of immunoaffinity-purified Cyt b by resonance energy transfer.