The adduct forming (bleaching) properties of four cationic triarylmethane dyes (methyl green, MeG(+); malachite green, MG(+); pararosaniline, PR+; crystal violet, CV+) were studied at 25 degreesC, in 100 mM MOPS buffer (pH 8), using simple nitrogen and sulfur nucleophiles (imidazole, 6-aminocaproic acid, 2-mercaptoethanol, reduced glutathione) or proteins (chicken ovalbumin, OA; human serum albumin, HSA; human gamma-globulins, IgG) as addends. Among simple nucleophiles, significant adduct formation was observed only with thiols. The apparent dissociation constants (K-d') at pH 8 for the 2-mercaptoethanol adducts of MeG(+), MG(+), PR+ and CV+ were 0.034, 0.22, 1.4 and 44 mM, respectively. Kd' values for the glutathione adducts of MeG(+) and MG(+) were 0.027 and 0.21 mM. Methyl and malachite green were the only dyes to be bleached by proteins at moderate concentrations (150 muM). Bleaching was multiphasic, summing contributions from multiple nucleophilic centers. In contrast to the trend in the reactions with simple nucleophiles, MeG(+) was generally more resistant to protein-mediated bleaching than MG(+): OA and HSA contributed 78 and 36%, respectively, to the total color loss in MG(+); the corresponding contributions to the bleaching of MeG(+) were 16 and 15%. With both dyes IgG-mediated bleaching amounted to ca. 30%. It appeared that protein-borne sulfhydryl groups could add to MG(+) but not to MeG(+). The inferior reactivity of MeG(+) towards protein-SH may arise from hindered access of this nucleophile to the central carbon of the TAM(+) nucleus. The exceptional tendency of MG(+) to add protein-SH needs to be accounted for. One possibility is that SH groups, excluded from the central carbon, add to the unsubstituted phenyl ring unique to MG(+). (C) 2003 Elsevier Ltd. All rights reserved.