Interaction of albumins and hemin with aromatic antioxidants: A spectrophotometric and fluorometric study

Difference spectrophotometry and fluorescence quenching of human and bovine serum albumins were used to determine their association constants(K _a) with hemin in buffered physiological saline (pH 7.4) supplemented with 2% dimethylsulfoxide or in 40% aqueous dimethylformamide (pH 7.4).K _a values depended on the medium, the extent of albumin delipidation, and on the method of determination. The formation of hemin complexes witho-phenylenediamine, tetramethylbenzidine, gallic acid, its polydisulfide, and two substituted di-tert-butyl pyrocatechols was studied by difference spectrophotometry in the same media;K _a values for the complexes were calculated and compared to each other. The formation of complexes of these aromatic ligands with albumins was studied fluorometrically;K _a values were of order of ∼10⁵ M⁻¹ and decreased with the ligand hydrophobicity.