title: Affinity purification and reconstitution of human phagocyte flavocytochrome B for detection of conformational dynamics in the membrane.
abstract: Human flavocytochrome b (Cyt b) is the core electron transferase of the NADPH oxidase in phagocytes and a number of other cell types. The oxidase complex generates superoxide, initiating production of a cascade of reactive oxygen species critical for the killing of infectious agents. Many fundamental questions still remain concerning its structural dynamics and electron transfer mechanisms. In particular, Cyt b structure/function correlates in the membrane have been relatively unstudied. In order to facilitate the direct analysis of Cyt b structural dynamics in the membrane, the following method provides rapid and efficient procedures for the affinity purification of Cyt b from isolated neutrophil membrane fractions and its functional reconstitution in purified lipid preparations. The protocol presented here contains some new optimized procedures that will facilitate Cyt b isolation and reconstitution. Additional methods are presented that facilitate examination of conformational dynamics of the membrane reconstituted purified Cyt b by fluorescence resonance energy transfer (FRET) as measured by steady-state and lifetime fluorescence techniques.
authors: Department of Microbiology, Montana State University, Bozeman, MT, USA.
authors: Marcia
authors: M
authors: Riesselman
authors: Algirdas J
authors: AJ
authors: Jesaitis
mesh_terms: D002846:Chromatography, Affinity / Q000379:methods; D003573:Cytochrome b Group / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D019255:NADPH Oxidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D010586:Phagocytes / Q000201:enzymology*; D010713:Phosphatidylcholines / Q000737:chemistry; D011487:Protein Conformation
chemical_list: D003573:Cytochrome b Group; D008051:Lipid Bilayers; D010713:Phosphatidylcholines; C036888:cytochrome b558; D019255:NADPH Oxidases