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Difference spectrophotometry and fluorescence quenching of human and bovine serum albumins were used to determine their association constants (Ka) with hemin in buffered physiological saline (pH 7.4) supplemented with 2% dimethylsulfoxide or in 40% aqueous dimethylformamide (pH 7.4). Ka values depended on the medium, the extent of albumin delipidation, and on the method of determination. The formation of hemin complexes with o-phenylenediamine, tetramethylbenzidine, gallic acid, its polydisulfide, and two substituted di-tert-butyl pyrocatechols was studied by difference spectrophotometry in the same media; Ka values for the complexes were calculated and compared to each other. The formation of complexes of these aromatic ligands with albumins was studied fluorometrically; Ka values were of order of approximately 10(-5) M-1 and decreased with the ligand hydrophobicity.

The substituted pyrocatechols sodium (2,3-dihydroxy-4,6-di-tert-butyl-phenyl)-S-thiosulfate (InH1) and 3-(S-glutathionyl)-4,6-di-tert-butyl-pyrocatechol (InH2) effectively inhibited oxidation of o-phenyle-nediamine and tetramethylbenzidine (TMB) catalyzed by hemin or its complex with BSA (methemalbumin). The method of competitive reactions of aromatic amines (PDA, TMB) and pyrocatechols (InH1, InH2) with active radicals was used for the quantitative determination of rates of radical initiation in systems containing hemin (methemalbumin) and H2O2 (TBHP). The use of the inhibitor method for heme-protein-peroxide systems is complicated by heterogeneity of the radicals generated (HO., RO., HO2., RO2., peroxide-like compounds I and II), formation of complexes of the inhibitors with protein component, and possible interaction of active radicals with organic co-solvents (DMF or DMSO). The system hemin (methemalbumin)-H2O2 (TBHP)-aromatic amines can be used for testing the efficacy of potential bioantioxidants.