Tryptophan 19 residue is the origin of bovine β-lactog1obu1in fluorescence

beta-Lactoglobulin consists of a single polypeptide of 162 amino acid residues with 2 Trp residues, Trp 19 present in a hydrophobic pocket and Trp 61 present at the surface of the protein near the pocket. This study aimed to characterize the respective contribution of the two Trp residues to the overall fluorescence of the protein. We added for that calcofluor white, an extrinsic fluorophore, which, at high concentration compared to that of the protein, quenches completely emission of hydrophobic Trp residue(s). The study was performed at different pH(s) by recording fluorescence steady state spectra and measuring fluorescence lifetimes of the Trp-residues using Single Time Photon Counting method. Our results indicate that addition of calcofluor white does not induce a red shift of the tryptophan(s) emission peak (332 nm) but only a decrease in the fluorescence intensity. This means that Trp 61 residue does not contribute to the protein emission, tryptophan emission occurs from Trp 19 residue only. Also, excitation spectrum peak position (283 nm) of beta-lactoglobulin is not modified upon calcofluor white binding. These results mean that structural rearrangements within beta-lactoglobulin are not occurring upon calcofluor white binding. Energy transfer between Trp 19 residue and calcofluor white occurs with 100% efficiency, i.e. the two fluorophores are very close one to each other (<5 A). This energy transfer is not Forster type. Fluorescence intensity decay of Trp 19 residue occurs with three lifetimes, equal to 0.48, 1.49 and 4.29 ns at pH 2 (monomeric state). Very close values were obtained at the different studied pHs (2-12) and where p-lactoglobulin is at different quaternary structure or present in solution in a mixture of dimers and monomers. Our data are interpreted as the results of emission occurring from different substructures of the tryptophan, reached at the excited state. The populations of these substructures characterized by the pre-exponential parameters of the fluorescence lifetimes are dependent on the microenvironment of the fluorophore and on the local protein structure. C), 2013 Elsevier B.V. All rights reserved.