The formation of colorless adducts by four cationic triarylmethane dyes (TAM(+)s), methyl green (MeG(+)), malachite green (MG(+)), pararosaniline (PR+), and crystal violet (CV+) was studied spectrophotometrically at 25 degrees C, in 50 mM 3-(N-morpholino)propanesulfonic acid (MOPS) buffer (pH 8), by monitoring the loss in TAM(+) color in the absence and presence of human serum proteins as potential addends. Unfractionated serum caused a rapid bleaching of MeG(+) and MG(+), while PR+ and CV+ were unaffected. Sephacryl S200 HR chromatographic screening of the serum revealed two composite peaks of MeG(+)-bleaching activity. The major peak (M-r range, 40,000-130,000) overlapped with and extended on either side of the albumin peak. The minor peak corresponding to ca. 10% of the total MeG(+)-bleaching capacity had M-r > 230,000. MG(+)-bleaching activity dominated the entire chromatographic profile and implicated a multitude of minority proteins with a high capacity to form colorless MG adducts. It is concluded that highly electrophilic TAM(+)s such as MeG(+) and MG(+) must be quantitatively trapped in the form of dye-protein adducts in biological fluids and that the primary in vivo effects (e.g. toxicity) of such dyes most likely arise from ligand-type effects on multiple protein targets. Mechanisms that call for unmodified TAM(+) structure (radical-mediated redox changes, DNA intercalation) may be more relevant to the in vivo impact of dyes such as PR+ and CV+ that have a lower tendency to form adducts. (c) 2004 Wiley Periodicals, Inc.