Tallmadge D H, Borkman R F
School of Chemistry, Georgia Institute of Technology 30332.
Photochem Photobiol. 1990 Mar;51(3):363-8. doi: 10.1111/j.1751-1097.1990.tb01723.x.
Aqueous buffer solutions of the lens protein bovine gamma-II crystallin were irradiated at 295 nm in the presence of dithiothreitol to determine the individual photolysis susceptibilities of the four tryptophan residues. Reverse-phase high performance liquid chromatography was utilized to compare the tryptic peptide maps before and after irradiation. Sequence analysis of collected tryptic peptides showed that the four tryptophans in calf gamma-II crystallin. TRP-42, TRP-68, TRP-131, and TRP-157 appeared in four distinct tryptic peptides. Fluorescence and absorption (diode array) monitoring of the eluting peptides allowed assessment of the changes in peptide absorbance and fluorescence following irradiation. Tryptophan fluorescence losses of (40 +/- 15)%, (17 +/- 4)%, (35 +/- 5)% and (15 +/- 4)% were observed for the peptides containing TRP-42, TRP-68, TRP-131 and TRP-157, respectively. Thus the four tryptophans in calf gamma-II crystallin did not all photolyze at the same rate. The rate differences are presumably related to the microenvironments of the individual tryptophan residues, and this is discussed in terms of the known crystal structure of calf gamma-II crystallin.
在二硫苏糖醇存在的情况下，对晶状体蛋白牛γ-II 晶状体蛋白的水性缓冲溶液在295 nm波长下进行辐照，以确定四个色氨酸残基各自的光解敏感性。利用反相高效液相色谱比较辐照前后的胰蛋白酶肽图。对收集到的胰蛋白酶肽进行序列分析表明，小牛γ-II 晶状体蛋白中的四个色氨酸。TRP-42、TRP-68、TRP-131和TRP-157出现在四个不同的胰蛋白酶肽中。对洗脱肽进行荧光和吸收（二极管阵列）监测，可以评估辐照后肽的吸光度和荧光变化。分别观察到含有TRP-42、TRP-68、TRP-131和TRP-157的肽的色氨酸荧光损失为(40±15)%、(17±4)%、(35±5)%和(15±4)%。因此，小牛γ-II 晶状体蛋白中的四个色氨酸并非都以相同的速率发生光解。速率差异可能与各个色氨酸残基的微环境有关，这将根据小牛γ-II 晶状体蛋白的已知晶体结构进行讨论。
