Castelli F, White H D, Forster L S
Department of Biochemistry, University of Arizona, Tucson 85721.
Biochemistry. 1988 May 3;27(9):3366-72. doi: 10.1021/bi00409a037.
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.
已通过时间相关单光子计数法测量了牙鳕小清蛋白中单个色氨酸的荧光寿命。在存在饱和钙（大于2摩尔/摩尔蛋白质）的情况下，荧光衰减准确地呈单指数形式，寿命为4.6纳秒（0.1M氯化钾、20mM硼酸盐、1mM二硫苏糖醇、20℃、pH9）。用乙二醇双（β-氨基乙醚）-N，N，N'，N'-四乙酸完全去除小清蛋白中的钙后，发射衰减变为双相，并且观察到第二个更快的衰减过程，其寿命为1.3纳秒，在350nm处约占荧光发射的18%。钙饱和形式的荧光发射不会被碘化物明显淬灭。相反，完全去除钙后，如将数据外推至无限碘化物浓度所示，荧光是完全可淬灭的。这些结果表明，去除钙后蛋白质中色氨酸残基对溶剂的可及性大幅增加。淬灭数据的斯特恩-沃尔默图是非线性的，表明无钙蛋白质存在不止一种可淬灭构象。寿命和淬灭结果与在钙结合位点部分占据时仅存在显著浓度的两种化学计量物种，脱辅基小清蛋白和小清蛋白-Ca2一致。
