Combined quantitative cytophotometric method for staining indolyl and sulfhydryl groups and protein

Sulfenylation with 2-nitrophenylsulfenyl chloride (NPS-Cl), which is specific for tryptophyl and cysteinyl residues in protein, was applied to quantitative histochemistry. By measurement of the absorbance values at 370 nm of sections stained with NPS-Cl, Beer-Lambert's law was found to hold for NPS staining. Treatment of NPS-stained sections with 2-mercaptoethanol (ME) (NPS-ME staining) resulted in sulfenylation of tryptophyl residues only. For determination of the amounts of tryptophyl and cysteinyl residues per unit of protein, protein staining with Coomassie Brilliant Blue (CB) was combined with NPS and NPS-ME staining. CB and NPS-CBB staining also followed Beer-Lambert's law. By measuring the absorbance values at 370 and 650 nm of doubly stained sections, the relative contents of tryptophyl and cysteinyl residues in various tissue proteins were calculated. This method will be useful for the investigation of changes in both protein amount and composition.