title: Lifetime and quenching of tryptophan fluorescence in whiting parvalbumin.
abstract: The fluorescence lifetime of the single tryptophan in whiting parvalbumin has been measured by time-correlated single-photon counting. In the presence of saturating calcium, greater than 2 mol/mol of protein, the decay of fluorescence is accurately single exponential with a lifetime of 4.6 ns (0.1 M KCl, 20 mM borate, 1 mM dithiothreitol, 20 degrees C, pH 9). Upon complete removal of calcium from parvalbumin with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid the emission decay becomes biphasic, and a second more rapid decay process with a lifetime of 1.3 ns comprising approximately 18% of the fluorescence emission at 350 nm is observed. The fluorescence emission of the calcium-saturated form is not measurably quenched by iodide. In contrast, upon complete removal of calcium, the fluorescence is completely quenchable as shown by extrapolation of the data to infinite iodide concentration. These results indicate that there is a large increase in the accessibility of the tryptophan residue in the protein to solvent upon removal of calcium. Stern-Volmer plots of the quenching data are nonlinear and indicate that there is more than one quenchable conformation of the calcium-free protein. The lifetime and quenching results are consistent with the presence of significant concentrations of only two stoichiometric species, apoparvalbumin and parvalbumin--Ca2, at partial occupancy of the calcium binding sites.
authors.affiliation: Department of Biochemistry, University of Arizona, Tucson 85721.
authors.forename: F
authors.initials: F
authors.lastname: Castelli
authors.forename: H D
authors.initials: HD
authors.lastname: White
authors.forename: L S
authors.initials: LS
authors.lastname: Forster
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D005399:Fishes; D007700:Kinetics; D009124:Muscle Proteins / Q000378:metabolism*; D010320:Parvalbumins / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence / Q000379:methods; D014364:Tryptophan
chemical_list: D009124:Muscle Proteins; D010320:Parvalbumins; D014364:Tryptophan; D002118:Calcium