The NADPH oxidase complex, responsible for reactive oxygen species (ROS) generation by phagocytes, consists of a membrane-associated flavocytochrome b 558 (a heterodimer of NOX2 and p22phox) and the cytosolic components p47phox, p67phox, Rac(1 or 2), and p40phox. NOX2 carries all redox stations through which electrons flow from NADPH to molecular oxygen, to generate the primary ROS, superoxide. For the electron flow to start, a conformational change in NOX2 is required. The dominant hypothesis is that this change is the result of the interaction of NOX2 with one or more of the cytosolic components (NADPH oxidase assembly). At the most basic level, assembly is the sum of several protein-protein interactions amon... More
The NADPH oxidase complex, responsible for reactive oxygen species (ROS) generation by phagocytes, consists of a membrane-associated flavocytochrome b 558 (a heterodimer of NOX2 and p22phox) and the cytosolic components p47phox, p67phox, Rac(1 or 2), and p40phox. NOX2 carries all redox stations through which electrons flow from NADPH to molecular oxygen, to generate the primary ROS, superoxide. For the electron flow to start, a conformational change in NOX2 is required. The dominant hypothesis is that this change is the result of the interaction of NOX2 with one or more of the cytosolic components (NADPH oxidase assembly). At the most basic level, assembly is the sum of several protein-protein interactions among oxidase components. This chapter describes a reductionist approach to the identification of regions in oxidase components involved in assembly. This approach consists of "transforming" one component in an array of overlapping synthetic peptides and assessing binding to the peptides of another component, represented by a recombinant protein. The peptides are tagged with biotin, at the N- or C-terminus, and immobilized on streptavidin-coated 96-well plates. The protein partners are expressed with a 6His tag and added to the plates in the fluid phase. Binding of the protein to the peptides is quantified by a kinetic ELISA , using a peroxidase-conjugated anti-polyhistidine antibody. Protein-peptide binding assays were applied successfully to (a) identifying the binding site on one component (represented by peptides) for another component (proteins), (b) precisely defining the "binding sequence," (c) acquiring information on the binding site in the partner protein, (d) investigating the effect of conformational changes in proteins on binding to peptides, (e) determining the effect of physicochemical modification of peptides on binding of proteins, and (f) identifying epitopes recognized by anti-oxidase component antibodies by binding of antibody to peptide arrays derived from the component.
