The resolution of heterogeneous fluorescence of multitryptophan-containing proteins studied by a...

From acrylamide quenching results, analyzed by an itterative non-linear least-squares method, we have shown that the fluorescence of multitryptophan-containing proteins, such as horse-liver alcohol dehydrogenase, 3-phosphoglycerate kinase and lysozyme, can be resolved for different segmental contributions, each characterized by collisional (Ki) and static (Vi) quenching constants. The ability to resolve the heterogeneous fluorescence of proteins makes it possible to follow changes in dynamics of the individual residues. In yeast 3-phosphoglycerate kinase, which contains only two tryptophan residues, three fluorescent fractions, characterized by different accessibility to the quencher, were observed. Two of them are assigned to one of the tryptophan residue. This may be interpreted in terms of conformational fluctuations, which facilitate the access of acrylamide molecules to the buried tryptophan residues.