Detection of nitric oxide radical and determination of its scavenging activity by antioxidants using spectrophotometric and spectrofluorometric methods

Abstract

Although reactive nitrogen species (RNS) may attack biomacromolecules and cause tissue damage when unbalanced by natural antioxidant defenses of the organism, they can also take part in cell signaling under different physiological states and defend against certain pathogens. Since there is a scarcity of analytical methods to detect radicalic (NO)-N-center dot and its scavengers, a functionalized gold nanoparticle-based spectrophotometric method and a spectrofluorometric method have been separately developed to test antioxidant activity toward scavenging of (NO)-N-center dot produced from sodium nitroprusside (SNP). The spectrophotometric method involves conversion of (NO)-N-center dot to nitrite, followed by the formation of an azo dye with 4-aminothiophenol (4-ATP)-modified gold nanoparticles (AuNPs) and N-(1-naphthyl)-ethylene diamine dichloride (NED) and its absorbance measurement at 565 nm. Calibration equations were established by taking the absorbance difference in the presence and absence of antioxidants. In the spectrofluorometric method, the excess of NO radicals, after being scavenged by thiol type antioxidants, caused a decrease in resorcinol fluorescence. The developed spectrophotometric method was applied to orange juice and its trolox equivalent (TE) antioxidant activity was found. By further applying the developed methods to real samples such as bovine serum albumin (BSA), fetal bovine serum (FBS), saliva and certain biomolecules, it is envisaged that these novel methods improving the selectivity of previous methods can be useful in human health and disease research associated with nitric oxide. The developed methods were compared and validated against the conventional Griess assay with Student t-test and F tests.