Background

This colorimetric nitric oxide assay kit employs immunoaffinity purified Nitrate Reductase enabling the measure of total nitric oxide (NO) produced in in vitro experimental systems. Nitric oxide can be spectrophotometrically assayed by measuring the accumulation of its stable degradation products, nitrate and nitrite. The ratio of these two products in biological fluids, tissue culture media, etc. may vary substantially. Hence, for accurate assessment of the total nitric oxide generated, one must monitor both nitrate and nitrite. An excellent solution to this problem is the enzymatic conversion of nitrate to nitrite by the enzyme nitrate reductase (NaR), followed by quantitation of nitrite using Griess Reagent. In addition to providing all necessary components in a microtiter format, this kit employs affinity purified nitrate reductase and NADH, thereby circumventing some of the potential problems reported for nitric oxide measurement using NADPH dependent nitrate reductases.
 

Assay Principle

In aqueous solution, nitric oxide rapidly degrades to nitrate and nitrite. Spectrophotometric quantitation of nitrite using Griess Reagent is straightforward, but does not measure nitrate. This kit employs the NADH-dependent enzyme nitrate reductase (NaR) for conversion of nitrate to nitrite prior to quantitation of nitrate using Griess reagent — thus providing for accurate determination of total NO production.

This colorimetric nitric oxide assay kit can be used to accurately measure as little as 1 pmol/μL (~1μM) NO produced in aqueous solutions. Very little sample is required (5 to 85 μL depending on the [NO] in the sample. The completed reaction is read at 540 nm.

References

1. Schmidt, H. H., et. al.; (1995) Biochemica 2:22-23