Interaction of nitric oxide with 2-thio-5-nitrobenzoic acid: Implications for the determination of free sulfhydryl groups by Ellman's reagent

Nitric oxide (NO) in an aerobic environment, reacts with the sulfhydryl groups of proteins to form nitroso thiols. Ellman's reagent, 5,5'- dithiobis(2-nitrobenzoic acid), DTNB, is widely used for the determination of -SH groups. In this procedure, DTNB, a symmetric aryl disulfide, reacts with the free thiol to give a mixed disulfide plus 2-nitro-5-thiobenzoic acid (TNB) which is quantified by its absorbance at 412 nm. We observed that the presence of NO during the determination of SH groups in a reaction system containing glutathione (GSH) or bovine serum albumin (BSA) plus DTNB resulted in an inhibition in the detection of TNB. Addition of NO donors or NO gas after TNB was already formed led to the bleaching of yellow color and loss of absorbance at 412 nm. These interactions did not occur under anaerobic conditions. Decreased formation of TNB therefore appeared to be due not only to destruction of SH groups of BSA or GSH by NO (S-nitrosation) and consequently to lower TNB formation, but also to direct reaction of NO/O₂ with TNB. The mechanism(s) of inhibition of accumulation of TNB by NO was evaluated. NO generated by DEA/NO, SNAP, or spermine/NO, as well as gaseous NO or BSA-NO, directly interacted with TNB, followed by decreased absorbance at 412 nm in a concentration- and time-dependent manner. Kinetics of NO/O₂ interaction with TNB were dependent on the ability of the NO donors to release NO as the donors with a short half-life bleached the yellow color of TNB faster. The requirement for O₂ suggests that nitrogen oxide or higher oxides of NO(x) are responsible for interaction with TNB. The UV/VIS spectrum of the final product formed during the interaction of NO with TNB was identical to that of DTNB. These results suggest that interaction of NO (NO(x)) with TNB resulted in the formation of an unstable nitrosothiol, followed by oxidation and dimerization back to the corresponding disulfide, DTNB. Therefore, determination of SH groups in proteins by Ellman's reagent after or in the presence of NO treatment is complicated since the reduced form of DTNB, TNB, can be reoxidized by NO back to DTNB, with subsequent loss of absorbance at 412 nm.