Two-dimensional fluorescence correlation spectroscopy: resolution of fluorescence of tryptophan residues in horse heart myoglobin.
Generalized two-dimensional (2D) fluorescence correlation spectroscopy has been used to resolve fluorescence of two tryptophan (Trp) residues in horse heart myoglobin. Fluorescence quenching is employed as a perturbation mode for causing intensity changes in the fluorescence (quenching perturbation). Two kinds of quenchers, iodide ion and acrylamide, are used for inducing fluorescence intensity change. This technique works because the Trp residue located at the 7th position (W7) is known to be easily accessible to the quencher, whereas that located at the 14th position (W14) is not. By this technique, the fluorescence spectra of the two Trp residues were clearly resolved. From asynchronous maps, it was also shown that the quenching of W7 fluorescence is brought about prior to the quenching of W14 fluorescence. This result is consistent with the structure of horse heart myoglobin that was proposed earlier. Furthermore, it was elucidated that the present 2D analysis is not interfered with by Raman bands of the solvents, which sometimes brings difficulty into conventional fluorescence analysis.
Nakashima
Kenichi
K
Department of Chemistry, Faculty of Science and Engineering, Saga University, Saga 840-8502, Japan. nakashik@cc.saga-u.ac.jp
Yuda
Kazuki
K
Ozaki
Yukihiro
Y
Noda
Isao
I
D000465:Algorithms; D000818:Animals; D006736:Horses; D009206:Myocardium / Q000737:chemistry*; D009211:Myoglobin / Q000032:analysis* / Q000737:chemistry*; D011487:Protein Conformation; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013223:Statistics as Topic; D014364:Tryptophan / Q000032:analysis* / Q000737:chemistry*
D009211:Myoglobin; D014364:Tryptophan