The effect of added protein on the nonphotochemical bleaching of malachite green cation (MG(+)) in aqueous solution (100 mM MOPS, pH 8, or 100 mM CAPS, pH 10; 25degreesC) was studied using chicken egg albumin (OA) and human serum albumin (HSA). Bleaching in the absence of protein (carbinol formation) was a pseudofirst-order process with k(0) = 0.012 +/- 0.002 and 0.025 +/- 0.001 min(-1) at pH 8 and 10, respectively. Progress curves in the presence of OA or HSA were multiphasic; an initially fast decrease in A(620) was followed by a slower decay which could be resolved into two protein-related, first-order components. Protein concentrations causing 50% reduction in carbinol formation via the solvolytic route were: [OA] 60 muM (pH 8) and 15 muM (pH 10); [HSA] = > 200 muM (pH 8) and 1.5 PM (pH 10) ([MG(+)](O) approximate to8.5 muM). The rate constants for the 2 OA-related components varied with both pH and protein concentration: At pH 8, [OA]=40-300 muM, k(1) =0.26-2.4 and k(2)=0.05-0.26 min(-1); at pH 10, [OA] 10-150 muM, k(1)=1.8-12 and k(2)=0-09 -2.6 min(-1). The k values for HSA did not show a systematic dependence on protein concentration: at pH 8, k(1)=0.62+/-0.19 and k(2)=0.16+/-0.09 min(-1); at pH 10, k(1)=3.3+/-0.50 and k(2)=0.32+/-0.10 min(-1). The reversibility of the protein-mediated bleaching process was tested by adjusting the pH to 4.5 with glacial acetic acid and monitoring the recovery in A620. Recovery in the presence of OA pointed to three leukodye-protein populations, reverting instantly, slowly (k = 0.003-0.006 min(-1)) or not at all (k < 10(-4) min(-1)). Recovery in the HSA-bleached samples was instantaneous and nearly quantitative. The results indicate that adduct formation between proteins and MG(+) can occur at a significant rate at moderate pH and that some proteins (exemplified by OA in this study) have a capacity to act as irreversible scavengers of the cationic dye. Such involvement of proteins in the nonphotochemical conversion of MG(+) to colorless forms may be a limiting factor in biomedical applications which rest on the availability of the cationic species. (C) 2002 Elsevier Science Ltd. All rights reserved.