Estimation of disulfide bonds using 2-nitro-5-thiosulfobenzoic acid: limitations

Recently an elegant method for the quantification of the number of disulfide bonds in proteins and peptides has been reported [T.W. Thannhauser, Y. Konishi, and H.A. Scheraga (1984) Anal. Biochem. 138, 181-188]. The method is based on the quantification of 2-nitro-5-thiobenzoate (NTB) formed from the reaction of 2-nitro-5-thiosulfobenzoate with disulfides in the presence of excess sodium sulfite. Here it is reported that the NTB anion undergoes photochemical reaction with excess sulfite in the system, which results in the rapid disappearance of absorbance at 412 nm in the presence of light. The nonchromophoric derivative of this photochemical reaction is tentatively identified as a sulfo derivative of NTB. Based on these observations it is suggested that, for the quantification of disulfide bonds using the NTSB method, the assay should be carried out in the dark.