Document Type

Article

Publication Date

5-15-2009

Publication Title

Talanta

Abstract

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 spectrophotometric assay based on the 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.

Comments

The authors wish to acknowledge financial support by the US DOE-BES through a grant of the “Catalysis Science” program to M.B. (DE-FG02-03ER15462). This research was partially supported by an FRD grant from CSU.

DOI

10.1016/j.talanta.2008.12.074

Version

Postprint

Volume

78

Issue

3

Download

Included in

Chemistry Commons

Share

COinS