title: Two-dimensional fluorescence correlation spectroscopy IV: resolution of fluorescence of tryptophan residues in alcohol dehydrogenase and lysozyme.
abstract: Generalized two-dimensional (2D) fluorescence correlation spectroscopy has been used to resolve the fluorescence spectra of two tryptophan (Trp) residues in alcohol dehydrogenase and lysozyme. In each protein, one Trp residue is buried in a hydrophobic domain of the protein matrix and the other Trp residue is located at a hydrophilic domain close to the protein-water interface. Fluorescence quenching by iodide ion, a hydrophilic quencher, was employed as a perturbation to induce the intensity change in the spectra. The Trp residue which is located at the hydrophilic domain is effectively quenched by the quencher, while the Trp residue located at the hydrophobic domain is protected from the quenching. Therefore, the fluorescence of these two Trp residues have a different sensitivity to the quenching, showing a different response to the concentration of the quencher. Fluorescence spectra of the two Trp residues in alcohol dehydrogenase, which are heavily overlapped in conventional one-dimensional spectra, have been successfully resolved by the 2D correlation technique. From the asynchronous correlation map, it was revealed that the quenching of Trp located at the hydrophobic part was brought about after that of Trp located at the hydrophilic part. In contrast, the fluorescence spectra of the two Trp residues could not be resolved after the alcohol dehydrogenase was denatured with guanidine hydrochloride. These results are consistent with the well-known structure of alcohol dehydrogenase. Furthermore, it was elucidated that the present 2D analysis is not interfered by Raman bands of the solvent, which sometimes bring difficulty into the conventional fluorescence analysis. Fluorescence spectra of the Trp residues in lysozyme could not be resolved by the 2D correlation technique. The differences between the two proteins are attributed to the fact that the Trp residue in the hydrophobic site of lysozyme is not sufficiently protected from the quenching.
authors.affiliation: Department of Chemistry, Faculty of Science and Engineering, Saga University, Saga 840-8502, Japan.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Fukuma
authors.forename: Kenichi
authors.initials: K
authors.lastname: Nakashima
authors.forename: Yukihiro
authors.initials: Y
authors.lastname: Ozaki
authors.forename: Isao
authors.initials: I
authors.lastname: Noda
mesh_terms: D000426:Alcohol Dehydrogenase / Q000737:chemistry*; D005453:Fluorescence; D009113:Muramidase / Q000737:chemistry*; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013056:Spectrophotometry, Ultraviolet; D014364:Tryptophan / Q000032:analysis*
chemical_list: D014364:Tryptophan; D000426:Alcohol Dehydrogenase; D009113:Muramidase