Pande J, McDermott M J, Callender R H, Spector A
Department of Ophthalmology, College of Physicians and Surgeons, Columbia University, New York 10032.
Arch Biochem Biophys. 1989 Feb 15;269(1):250-5. doi: 10.1016/0003-9861(89)90106-9.
Laser Raman spectroscopy has been applied to native and dithiothreitol-treated bovine cortical gamma II crystallin to examine the state of the thiol groups and the presence of a putative disulfide bridge. The data reveal significant differences in two key spectral regions. In the thiol stretching region (2500-2600 cm-1), the dithiothreitol-reduced form shows a 25% increase in the integrated Raman signal as compared to the native form. The magnitude of this increase corresponds to the presence of 1 mol of disulfide/mol of gamma II as determined both by the Raman data and the previous biochemical analysis from this laboratory. In the disulfide stretching region (500-540 cm-1), the native form shows a line near 511 cm-1 which is absent in the reduced form. Both native and reduced forms show a triple-banded thiol signal with one or more distinct shoulders, suggesting at least three and perhaps five different environments for the cysteine residues. The difference spectrum, obtained by a 1:1 computer subtraction of the native from the reduced form, indicates that the increase in thiol signal is centered around 2572 cm-1. In every other spectral region, both native and reduced gamma II forms are closely similar. These results strongly support the biochemical data reported earlier and indicate that the reduction of the single disulfide bridge is accompanied by minimal changes in secondary structure in solution.
激光拉曼光谱已应用于天然的和经二硫苏糖醇处理的牛晶状体皮质γII晶状体蛋白，以检测硫醇基团的状态和假定的二硫键的存在。数据显示在两个关键光谱区域存在显著差异。在硫醇伸缩区域（2500 - 2600 cm-1），与天然形式相比，经二硫苏糖醇还原的形式的拉曼信号积分增加了25%。这种增加的幅度与每摩尔γII存在1摩尔二硫键相对应，这是通过拉曼数据和本实验室先前的生化分析确定的。在二硫键伸缩区域（500 - 540 cm-1），天然形式在511 cm-1附近显示出一条谱线，而还原形式中不存在。天然形式和还原形式均显示出具有一个或多个明显肩峰的三带硫醇信号，这表明半胱氨酸残基至少处于三种，可能五种不同的环境中。通过将还原形式与天然形式以1:1的比例进行计算机相减得到的差谱表明，硫醇信号的增加集中在2572 cm-1附近。在其他每个光谱区域，天然的和还原的γII形式都非常相似。这些结果有力地支持了先前报道的生化数据，并表明单一二硫键的还原伴随着溶液中二级结构的最小变化。
