Kenoth Roopa, Swamy Musti J
School of Chemistry, University of Hyderabad, India.
J Photochem Photobiol B. 2003 Mar;69(3):193-201. doi: 10.1016/s1011-1344(03)00021-6.
Steady-state and time-resolved fluorescence spectroscopic studies have been carried out on Trichosanthes cucumerina seed lectin (TCSL). The fluorescence emission maximum of TCSL in the native state as well as in the presence of 0.1 M lactose is centered around 331 nm, which shifts to 347 nm upon denaturation with 8 M urea, indicating that all the tryptophan residues of this protein in the native state are in a predominantly hydrophobic environment. The exposure and accessibility of the tryptophan residues of TCSL and the effect of ligand binding on them were probed by quenching studies employing two neutral quenchers (acrylamide and succinimide), an anionic quencher (I(-)) and a cationic quencher (Cs(+)). Quenching was highest with acrylamide and succinimide with the latter, which is bulkier, yielding slightly lower quenching values, whereas the extent of quenching obtained with the ionic quenchers, I(-) and Cs(+) was significantly lower. The presence of 0.1 M lactose led to a slight increase in the quenching with acrylamide and iodide, whereas quenching with succinimide and cesium ion was not significantly affected. When TCSL was denatured with 8 M urea, both acrylamide and succinimide yielded upward-curving Stern-Volmer plots, indicating that the quenching mechanism involves both dynamic and static components. Quenching data obtained with I(-) and Cs(+) on the urea-denatured protein suggest that charged residues could be present in close proximity to some of the Trp residues. The Stern-Volmer plots with Cs(+) yielded biphasic quenching profiles, indicating that the Trp residues in TCSL fall into at least two groups that differ considerably in their accessibility and/or environment. In time-resolved fluorescence experiments, the decay curves could be best fit to biexponential patterns, with lifetimes of 1.78 and 4.75 ns for the native protein and 2.15 and 5.14 ns in the presence of 0.1 M lactose.
已对栝楼种子凝集素（TCSL）进行了稳态和时间分辨荧光光谱研究。天然状态以及存在0.1 M乳糖时，TCSL的最大荧光发射集中在331 nm左右，在用8 M尿素变性后移至347 nm，这表明该蛋白质天然状态下的所有色氨酸残基主要处于疏水环境中。通过使用两种中性猝灭剂（丙烯酰胺和琥珀酰亚胺）、一种阴离子猝灭剂（I(-)）和一种阳离子猝灭剂（Cs(+)）的猝灭研究，探究了TCSL色氨酸残基的暴露和可及性以及配体结合对它们的影响。丙烯酰胺和琥珀酰亚胺的猝灭作用最强，后者体积更大，猝灭值略低，而离子猝灭剂I(-)和Cs(+)的猝灭程度明显较低。0.1 M乳糖的存在导致丙烯酰胺和碘化物的猝灭略有增加，而琥珀酰亚胺和铯离子的猝灭没有受到显著影响。当用8 M尿素使TCSL变性时，丙烯酰胺和琥珀酰亚胺都产生向上弯曲的Stern-Volmer图，表明猝灭机制涉及动态和静态成分。用I(-)和Cs(+)对尿素变性蛋白获得的猝灭数据表明，带电残基可能与一些色氨酸残基紧邻。Cs(+)的Stern-Volmer图产生双相猝灭曲线，表明TCSL中的色氨酸残基至少分为两组，它们的可及性和/或环境有很大差异。在时间分辨荧光实验中，衰减曲线最适合双指数模式，天然蛋白的寿命为1.78和4.75 ns，在存在0.1 M乳糖时为2.15和5.14 ns。
