Reductive decomposition of a diazonium intermediate by dithiothreitol affects the determination of NOS turnover rates

TALANTA

卷:78

Article

Perera, Indika N.¹  Sabbagh, Talal¹  Boutros, Jean A.¹  Bayachou, Mekki¹ 

[1] Cleveland State Univ, Dept Chem SR 397, Cleveland, OH 44115 USA

关键词: Griess assay;    Diazonium;    Dithiothreitol;    Nitric oxide synthase;    Turnover;    External calibration;    Standard addition;   

DOI  :  10.1016/j.talanta.2008.12.074

来源: Elsevier

【 摘 要 】

Accurate determination of nitrite either as Such or as the breakdown product of nitric oxide (NO) is critical in a host of enzymatic reactions in various settings addressing structure-function relationships, as well as mechanisms and kinetics of molecular operation of enzymes. The most common way to quantify nitrite, for instance in nitric oxide synthase (NOS) mechanistic investigations, is the spectro photo metric assay based on (lie Griess reaction through external standard calibration. This assay is based on a two-step diazotization reaction, in which a cationic diazonium derivative of sulfanilamide is formed as intermediate before the final absorbing azo-product. We show that this intermediate is very sensitive to reducing agents that may be transferred from the reaction media under investigation. The interaction of this vital intermediate with the reducing agent, dithiothreitol (DTT), which is widely used in NOS reactions, is characterized by both electrochemical and spectroscopic means. The effect of DTT on the performance of external calibration, both in sample recovery studies and in actual NOS reactions, is presented. Finally an alternative method of standard additions, which partially compensates for the accuracy and sensitivity problems of external calibration, is proposed and discussed. (C) 2009 Elsevier B.V. All rights reserved.

【 授权许可】

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