Englander J J, Downer N W, Englander S W
J Biol Chem. 1982 Jul 25;257(14):7982-6.
The hydrogen exchange behavior of rhodopsin was re-examined by studies of the protein in the disc membrane and after solubilization in octyl glucoside. The methods used measure either the peptide hydrogens alone (hydrogen-deuterium exchange by infrared spectroscopy) or all slowly exchanging hydrogens (hydrogen-tritium exchange by hel filtration). Under mild exchange conditions, disc membranes and solubilized lipid-free proteins show very similar exchange behavior, indicating the absence of slowly exchanging lipid protons. At high temperature, exchange of an additional large group of very slow peptide NH can be detected. The total number of slow hydrogens significantly exceeds the amide content, and apparently includes slowly exchanging protons from perhaps 40% of the protein's non-amide side chains. This is thought to require the involvement of many polar side chains in internal H-bonding. The exchange rates of the non-amide side chains sites have not been determined. However, to the extent that these contribute to the fast time region of the measured kinetic H-exchange curve, previously identified with exposed, non-H-bonded peptides, the estimate of freely exposed rhodopsin peptides must be reduced. The fraction of free peptides could range from a remarkably high value of 70% down to about 45%.
通过对盘膜中的视紫红质以及在辛基葡糖苷中溶解后的视紫红质进行研究，对视紫红质的氢交换行为进行了重新审视。所使用的方法要么单独测量肽氢（通过红外光谱进行氢-氘交换），要么测量所有缓慢交换的氢（通过凝胶过滤进行氢-氚交换）。在温和的交换条件下，盘膜和溶解的无脂蛋白质表现出非常相似的交换行为，这表明不存在缓慢交换的脂质质子。在高温下，可以检测到另外一大组非常缓慢的肽NH的交换。缓慢氢的总数明显超过酰胺含量，显然包括来自蛋白质约40%的非酰胺侧链的缓慢交换质子。这被认为需要许多极性侧链参与内部氢键形成。尚未确定非酰胺侧链位点的交换速率。然而，就这些位点对测量的动力学氢交换曲线的快速时间区域有贡献而言，此前确定为暴露的、非氢键结合的肽，对视紫红质自由暴露肽的估计必须降低。自由肽的比例可能从高达70%的显著值降至约45%。
