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

• Type: Article in International Scientific Journal
• Date: 2014

Authors

Simone M Marques (Author) Other; Joaquim C G E Esteves da Silva (Author) FCUP

Journal

Title: Analytical Methods

Vol. 6

Pages: 3741-3750

ISSN: 1759-9660

Publisher: Royal Society of Chemistry

Indexing

ISI Web of Knowledge - 2 Citations

Scopus - 2 Citations

Scientific classification

FOS: Agrarian Sciences > Other Agrarian Sciences

Other information

Authenticus ID: P-009-GPA

DOI: 10.1039/c4ay00317a

Abstract (EN): A novel optimized coupled bioluminescent assay for nitrogen monoxide free radical (nitric oxide, (NO)-N-center dot), 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)-N-center dot 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)-N-center dot donor, the assay was optimized through statistical experimental design methodology, namely Plackett-Burman (screening) and Box-Behnken (optimization) designs. The optimized method requires 5 mu L of sample per tube in a final reaction volume of 100 mu L. It is linear in the range from 10 to 100 nM of (NO)-N-center dot, 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.

Language: English

Type (Professor's evaluation): Scientific

No. of pages: 10