Marquezin Cássia Alessandra, Hirata Izaura Yoshico, Juliano Luiz, Ito Amando Siuiti
Instituto de Física da Universidade de São Paulo, SP, Brasil.
Biopolymers. 2003;71(5):569-76. doi: 10.1002/bip.10535.
We present results of time resolved fluorescence measurements performed in Tryptophan (Trp) derivatives and Trp-containing peptides in the pH range 3.0-11.0. For each compound a set of decay profiles measured in a given range of pH values was examined as a whole, using the global analysis technique. The data were fitted to two or three lifetime components and the analysis allowed the monitoring of the changes in the concentration of the different species contributing to the total fluorescence in that pH interval. The decay components were sensitive to the ionization state of groups neighboring the indol ring, and pK values for the equilibrium between protonated and deprotonated species were obtained from the preexponential factor of the lifetime components. In Trp, protonation of the amino terminal of the rotamer having electron transfer rate comparable to fluorescence decay rates was responsible for the interconvertion of a long lifetime component, to the 2.9 ns component usually observed in neutral pH. Trpbond;X peptides also have a single rotamer dominating the decay that is quenched by NH(3) (+). X-Trp peptides seem to be conformationally less restricted, and it is possible that rotamers interconvertion occur in high pH, increasing the population of nonquenched rotamers. Interconvertion between rotameric conformations of Trp are also present in the titration of ionizable groups in the side chain of peptides like His-Trp and Glu-Trp and control of pH is essential to the correct interpretation of fluorescence data in the study of peptides having such groups near to the Trp residue.
我们展示了在3.0 - 11.0的pH范围内对色氨酸（Trp）衍生物和含Trp肽进行时间分辨荧光测量的结果。对于每种化合物，使用全局分析技术对在给定pH值范围内测量的一组衰减曲线进行整体检查。数据拟合为两个或三个寿命成分，该分析允许监测在该pH区间内对总荧光有贡献的不同物种浓度的变化。衰减成分对吲哚环附近基团的电离状态敏感，并且从寿命成分的前指数因子获得质子化和去质子化物种之间平衡的pK值。在Trp中，具有与荧光衰减速率相当的电子转移速率的旋转异构体的氨基末端质子化导致长寿命成分向通常在中性pH下观察到的2.9 ns成分的相互转化。Trpbond;X肽也有一个主导衰减的单一旋转异构体，该异构体被NH(3) (+)淬灭。X - Trp肽似乎在构象上限制较少，并且在高pH下可能发生旋转异构体的相互转化，增加未淬灭旋转异构体的数量。在His - Trp和Glu - Trp等肽侧链中可电离基团的滴定中也存在Trp旋转异构体构象之间的相互转化，并且在研究Trp残基附近有此类基团的肽时，控制pH对于正确解释荧光数据至关重要。
