Adduct-forming tendencies of cationic triarylmethane dyes with proteins: Metabolic and toxicological implications

The formation of colorless adducts by four cationic triarylmethane dyes (TAM⁺s), methyl green (MeG⁺), malachite green (MG+), pararosani-line (PR⁺), and crystal violet (CV⁺) was studied spectrophotometrically at 25°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 Mr > 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.