A study of the adsorption of dyes on bovine serum albumin by the method of polarization of fluorescence

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Abstract
A method isdescr. for studying the adsorption of fluorescent dyes by macromolecules using measurements of the polarization and intensity of the fluorescent light. The method is applied to the adsorption of dyes by bovine serum albumin. The effect of pH on the adsorption is discussed. Negatively charged dye ions are adsorbed even when the net charge on the protein is negative. A positive charge on the dye molecule inhibits combination on either side of the isoelectric point. Basic dyes are adsorbed as the free base but not as the positive ion. Free carboxyl, sulfonic, hydroxyl or amino groups in the dye are not necessary for combination between dye and protein to occur. Br atoms increase the adsorption. Addition of related but non-fluorescent compounds can decrease the adsorption of the fluorescent dye by competition. Competition is also found between the dye and a variety of simple inorganic and organic ions. The activity of univalent negative inorganic ions is in the order of the Hofmeister series and that of univalent negative organic ions in the order of surface activity. Positive ions do not compete with the dyes. Characteristic changes in the absorption spectra and in the fluorescence of the dyes occur on adsorption. These changes can be reproduced if the dyes are adsorbed by cetyltrimethylammonium Br micelles or dissolved in 90% dioxane. The significance of these results is discussed and a comparison made between the adsorbed dye and the dye coupled covalently to serum albumin by the methods of Weber (Biochem. Jour. 51: 155. 1952). The results of this work can best be explained by the presence in serum albumin of a few surface regions where a local excess of positively charged amino acid residues is backed by residues with non-polar side chains,.