Neutrophil pp 325-411 | Cite as

Cell-Free NADPH Oxidase Activation Assays: A Triumph of Reductionism

  • Edgar PickEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2087)


The superoxide (O2·−)-generating NADPH oxidase complex of phagocytes comprises a membrane-associated heterodimeric flavocytochrome, known as cytochrome b558 (consisting of NOX2 and p22phox) and four cytosolic regulatory proteins, p47phox, p67phox, p40phox, and the small GTPase Rac. Under physiological conditions, in the resting phagocyte, O2·− generation is initiated by engagement of membrane receptors by a variety of stimuli, followed by signal transduction sequences leading to the translocation of the cytosolic components to the membrane and their association with the cytochrome, a process known as NADPH oxidase assembly. A consequent conformational change in NOX2 initiates the electron flow along a redox gradient, from NADPH to molecular oxygen (O2), leading to the one-electron reduction of O2 to O2·−. Historically, methodological difficulties in the study of the assembled complex derived from stimulated cells, due to its lack of stability, led to the design of “cell-free” systems (also known as “broken cells” or in vitro systems). In a major paradigm shift, the cell-free systems have as their starting point NADPH oxidase components derived from resting (unstimulated) phagocytes, or as in the predominant method at present, recombinant proteins representing the components of the NADPH oxidase complex. In cell-free systems, membrane receptor stimulation and the signal transduction sequence are absent, the accent being placed on the actual process of assembly, all of which takes place in vitro. Thus, a mixture of the individual components of the NADPH oxidase is exposed in vitro to an activating agent, the most common being anionic amphiphiles, resulting in the formation of a complex between cytochrome b558 and the cytosolic components and O2·− generation in the presence of NADPH. Alternative activating pathways require posttranslational modification of oxidase components or modifying the phospholipid milieu surrounding cytochrome b558. Activation is commonly quantified by measuring the primary product of the reaction, O2·−, trapped immediately after its generation by an appropriate acceptor in a kinetic assay, permitting the calculation of rates of O2·− production, but numerous variations exist, based on the assessment of reaction products or the consumption of substrates. Cell-free assays played a paramount role in the identification and characterization of the components of the NADPH oxidase complex, the performance of structure–function studies, the deciphering of the mechanisms of assembly, the search for inhibitory drugs, and the diagnosis of various forms of chronic granulomatous disease (CGD).

Key words

Reactive oxygen species Superoxide NADPH oxidase Cytochrome b558 NOX2 NOXes Cytosolic components p47phox p67phox Rac Cell-free assays Anionic amphiphile Arachidonic acid GTP Kinetic assay Superoxide dismutase Prenylation “Peptide walking” 



The research described in this chapter was supported by the Julius Friedrich Cohnheim-Minerva Center for Phagocyte Research, the Ela Kodesz Institute of Host Defense against Infectious Diseases, Israel Science Foundation Grants 428/01, 19/05, 49/09, 300/13, and 144/17, the Roberts-Guthman Chair in Immunopharmacology, the Walter J. Levy Benevolent Trust, the David Roberts Fund, and the Joseph and Shulamit Salomon Fund. It is important to point out that the cell-free system was discovered almost simultaneously by several investigators: R.A. Heyneman and R.E. Vercauteren, in Belgium, and Linda McPhail and John Curnutte, in the USA. All these investigators, independently, contributed greatly to the birth of the “cell-free” paradigm. Edgar Pick would like to thank the many postdoctoral fellows, students, and assistants, who over a period of close to four decades, were responsible for this considerable body of work. Among the many, Ms. Yael Bromberg, deserves special thanks for her dedication and perseverance up to and after the moment when we first saw cytochrome c being reduced by a homogenate of resting macrophages. Thanks go to my fellow scientists, too many to name, who provided materials and invaluable advice, for making this work possible. There is no greater satisfaction than the realization of the fact that, on so many occasions, what started as collaboration (called “networking,” these days) or competition, evolved into long-lasting friendships. Finally, no hard feelings are left toward the reviewers who rejected one of our papers describing the cell-free system and toward those who labeled the cell-free system an “in vitro artefact,” of no relevance to what happens in vivo. Time is a great healer.


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© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Department of Clinical Microbiology and Immunology, Sackler School of MedicineTel Aviv UniversityTel AvivIsrael

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