Spectrophotometric activity microassay for pure and recombinant cytochrome P450-type nitric oxide reductase

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dc.contributor.author Garny, Seike
dc.contributor.author Verschoor, J.A. (Jan Adrianus), 1953-
dc.contributor.author Gardiner, Neil
dc.contributor.author Jordaan, Justin
dc.date.accessioned 2014-02-03T09:08:27Z
dc.date.available 2014-02-03T09:08:27Z
dc.date.issued 2014-02
dc.description.abstract Nitric oxide reductase (NOR) of the P450 oxidoreductase family accepts electrons directly from its cofactor, NADH, to reduce two nitric oxide (NO) molecules to one nitrous oxide molecule and water. The enzyme plays a key role in the removal of radical NO produced during respiratory metabolism, and applications in bioremediation and biocatalysis have been identified. However, a rapid, accurate, and sensitive enzyme assay has not yet been developed for this enzyme family. In this study, we optimized reaction conditions for the development of a spectrophotometric NOR activity microassay using NOC-5 for the provision of NO in solution. We also demonstrate that the assay is suitable for the quantification and characterization of P450-type NOR. The Km and kcat kinetic constants obtained by this assay were comparable to the values determined by gas chromatography, but with improved convenience and cost efficiency, effectively by miniaturization. To our knowledge, this is the first study to present the quantification of NOR activity in a kinetic microassay format. en_US
dc.description.librarian hb2014 en_US
dc.description.sponsorship A CSIR parliamentary grant (Pretoria, South Africa) en_US
dc.description.uri http://www.elsevier.com/locate/yabio en_US
dc.identifier.citation Garny, S, Verschoor, JA, Gardiner, N & Jordaan, J 2014, 'Spectrophotometric activity microassay for pure and recombinant cytochrome P450-type nitric oxide reductase', Analytical Biochemistry, vol. 447, no.1, pp. 23-29. en_US
dc.identifier.issn 0003-2697 (print)
dc.identifier.issn 1096-0309 (online)
dc.identifier.other 10.1016/j.ab.2013.11.005
dc.identifier.uri http://hdl.handle.net/2263/33204
dc.language.iso en en_US
dc.publisher Elsevier en_US
dc.rights © 2013 Elsevier Ltd. All rights reserved. Notice : this is the author’s version of a work that was accepted for publication in Analytical Biochemistry. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Analytical Biochemistry, vol. 447, no.1, pp. 23-29, 2014. doi : 10.1016/j.ab.2013.11.005 en_US
dc.subject Enzyme assay en_US
dc.subject NADH en_US
dc.subject Nitric oxide reductase (NOR) en_US
dc.title Spectrophotometric activity microassay for pure and recombinant cytochrome P450-type nitric oxide reductase en_US
dc.type Postprint Article en_US


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