A nitric oxide quantitative assay by a glyceraldehyde 3-phosphate dehydrogenase/phosphoglycerate kinase/firefly luciferase optimized coupled bioluminescent assay

Abstract

A novel optimized coupled bioluminescent assay for nitrogen monoxide free radical (nitric oxide, ˙NO), an important environmental and physiological molecule, is presented. The method is based on the reaction catalyzed by glyceraldehyde 3-phosphate dehydrogenase (GAPDH), whose product is used as a substrate for phosphoglycerate kinase (PGK), generating adenosine 5′-triphosphate (ATP), which is an essential cofactor for the firefly luciferase bioluminescent reaction. Inhibition of GAPDH by ˙NO hampers the coupled reactions, leading to a depletion of ATP and hence a decrease in the bioluminescent signal. Using diethylamine NONOate (DEA-NONOate) as the ˙NO donor, the assay was optimized through statistical experimental design methodology, namely Plackett–Burman (screening) and Box–Behnken (optimization) designs. The optimized method requires 5 μL of sample per tube in a final reaction volume of 100 μL. It is linear in the range from 10 to 100 nM of ˙NO, with limits of detection and quantitation of 4 and 15 nM, respectively. Limitations in its application to biological samples, together with approaches to solve them, are discussed using human whole saliva and microalgae culture medium as examples.