The photobinding between riboflavin and the Trp residues from human and bovine serum albumins at two pH-dependent protein conformations was studied. At pH 7.0 both proteins showed photo-adduct formation with hyperbolic kinetics. In the bovine serum albumin this is attributed to the different locations of the two Trp residues. In the case of the human serum albumin, which has only one Trp residue, this behaviour may be related to different molecular conformations of the protein, as is also manifest in the iodide quenching experiments. At pH 3.5, the kinetics of the photo-adduct formation were found to be slower and showed a monophasic behaviour. These results are due to the conformational change of these proteins at acidic pH; the Trp residues of both proteins being now located in a more hydrophobic environment. When bovine serum albumin was anaerobically irradiated at pH 7.0 in the presence of 14C-riboflavin and then cleaved by CNBr, two peptides were obtained, containing the Trp-134 and Trp-212 residues, respectively. The incorporation of 14C-riboflavin in these samples was significantly higher at the level of the peptide containing the Trp-134 residue. Furthermore, it was demonstrated, that the energy transfer from enzymatically generated triplet acetone to riboflavin can also promote the binding of this vitamin to the Trp residues of human and bovine serum albumins.

The rate constants for the photodynamic inactivation of hen egg-white lysozyme at different temperatures were studied. Arrhenius plots of the methylene blue sensitized photo-inactivation of lysozyme gave an experimental activation energy of 7.5 kcal/mol. The rate constants for the photodynamic inactivation of lysozyme in the presence of riboflavin decreased almost linearly in the temperature range 4-38 degrees C. The photosensitized oxidation of lysozyme at -20 degrees C in freezing and non-freezing solvents was possible only in the presence of riboflavin. The effect of dye concentration on the quantum yield and rate constant for the photodynamic inactivation of lysozyme was examined. The quantum yields were lower when the concentrations of methylene blue used were low, and increased on increasing dye concentration, getting to a maximum and then declined at higher dye concentrations. It was found that in the case of riboflavin sensitized photo-inactivation of lysozyme both the rate constant and the quantum yield increased as the dye concentration increased. No maximum was observed over the range of dye concentrations studied. A new mechanism is postulated for the photodynamic action of lysozyme in the presence of riboflavin.

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We describe here the development of monoclonal antibodies to the hapten tryptophan-riboflavin, generated by irradiation of a solution of bovine serum albumin in the presence of riboflavin. The specificity of the three obtained monoclonal antibodies, named 1E6, 5H5, 5A8 all belonging to the IgG1 isotype, was assessed by a competitive enzyme-linked immunosorbent assay in the presence of an increasing concentration of the tryptophan-riboflavin adduct, obtained from an irradiated riboflavin-sensitized tryptophan solution. It was demonstrated that the tryptophan-riboflavin antibodies react with the soluble proteins of the eye lens; this reaction was more intense in the old rat lenses as compared to the young ones, and a maximum binding of the antibodies was obtained with the soluble protein fraction from the human cataractous lens. By indirect immunofluorescence, a reactivity associated with the protein matrix, localized in the lens central zone, was observed. In the peripheral zone of the lens, where the younger cells are found, a marked immunofluorescent emission was observed on structures preferentially localized in the nuclei.

The effect of iodide ion on the tryptophyl fluorescence of the homologous proteins lysozyme and alpha-lactalbumin in their native form, as well as in their modified structures and in fragments from these proteins was studied. By assessing the contribution to the total fluorescence of the exposed and buried Trp residues, and of the respective fluorescence quantum yields, the quantification of the number of Trp exposed to the solvent for all the species studied was possible. Both native proteins show an important increase in the number of Trp residues exposed to the solvent when treated with denaturing agents. The peptides L-II (aa 13-105) and alpha-I (aa 1-90) from lysozyme and alpha-lactalbumin, respectively, showed Trp residues with different degree of exposure, whereas the smaller fragments, L-III (aa 106-129) and alpha-II (aa 91-123), had all their Trp residues exposed to the solvent.