Spectrophotometric Determination of Nitrites in Biological Samples Using 1,2-Diaminoanthraquinone. Potential Application to the Determination of Nitric Oxide Synthase Activity

Abstract

The present article describes a new method for determining nitrite. The method is based on the reaction of 1,2-diaminoanthraquinone (DAQ) to form a colorless anthraquinone triazole. Absorbance was seen to change linearly with increasing nitrite concentrations in the (0.05–50 µM) range. In order to test the potential use of this method for the determination of nitric oxide synthase (NOS) activity, a panel of enzyme substrates (NADPH), cofactors (FMNH₂, FADH₂), reductants (DTT), and exogenous proteins were tested as potentially interfering substances. Under the experimental conditions used no single compound among those tested interfered in the assay. This is a clear advantage with respect to the Griess method, which shows interferences with NADPH, but more especially with respect to the DAN method, which presents interferences with NADPH, FMNH₂, FADH₂, and hemoglobin. Nevertheless, 1,2-diaminoanthraquinone is less water-soluble than DAN and the Griess reagent, making the use of this substrate difficult in the determination of nitric oxide synthase activity in biological samples. We propose to use this method for nonbiological samples or for environmental analysis.

Keywords:

• Nitrite
• Nitric oxide
• Nitric oxide synthase
• 1,2-Diaminoanthraquinone
• Spectrophotometric assay