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.
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Riesselman, M., Jesaitis, A.J. (2014). Affinity Purification and Reconstitution of Human Phagocyte Flavocytochrome b for Detection of Conformational Dynamics in the Membrane. In: Quinn, M., DeLeo, F. (eds) Neutrophil Methods and Protocols. Methods in Molecular Biology, vol 1124. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-845-4_24
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DOI: https://doi.org/10.1007/978-1-62703-845-4_24
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Publisher Name: Humana Press, Totowa, NJ
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Online ISBN: 978-1-62703-845-4
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