Sadler P J, Tucker A
Christopher Ingold Laboratories, Birkbeck College London, England.
Eur J Biochem. 1992 Apr 15;205(2):631-43. doi: 10.1111/j.1432-1033.1992.tb16821.x.
A variety of one- and two-dimensional 1H-NMR methods have been applied to the study of defatted 66.5-kDa bovine serum albumin in solution. 1. The majority of the protons gave rise to broad unresolved resonances and spectral enhancement methods for one-dimensional spectra were investigated in detail. A combination of exponential and sine-bell functions was particularly effective. 2. The presence of contaminating glycoproteins in some commercial samples of bovine serum albumin was readily detectable from their N-acetyl resonances at about 2.1 ppm. 3. The release of bound Cys (from mixed disulphide at Cys34) was observed after addition of dithiothreitol. 4. Through the use of two-dimensional shift-correlated spectroscopy, assignments of some 80 spin systems to amino acid type were made. 5. The pKa of the N-terminal Asp was measured as 7.8 (0.1 M phosphate buffer, 310 K). 6. 1H NMR spectra of bovine, human, porcine and rat serum albumins have been compared. Using sequence comparisons, specific assignments have been made for the N-terminal residues of bovine (Asp-Thr-His), human (Asp-Ala-His), porcine (Asp-Thr-Tyr) and rat (Glu-Ala-His) albumins, and for Thr189, Tyr155 and His59/377 of bovine albumin. 7. These NMR data suggest that certain local regions of bovine serum albumin are highly mobile yet structured in solution, and demonstrate that the application of both one- and and two-dimensional NMR methods will allow more detailed investigations of structural transitions in serum albumins induced by, for example, pH, drug and metal binding.
多种一维和二维¹H-NMR方法已被应用于研究溶液中脱脂的66.5 kDa牛血清白蛋白。1. 大多数质子产生了宽的未分辨共振峰，对一维谱的谱增强方法进行了详细研究。指数函数和正弦钟函数的组合特别有效。2. 从牛血清白蛋白一些商业样品中约2.1 ppm处的N-乙酰基共振峰很容易检测到污染糖蛋白的存在。3. 添加二硫苏糖醇后观察到结合的半胱氨酸（来自Cys34处的混合二硫键）的释放。4. 通过使用二维位移相关光谱，对约80个自旋系统进行了氨基酸类型的归属。5. 测定N端天冬氨酸的pKa为7.8（0.1 M磷酸盐缓冲液，310 K）。6. 比较了牛、人、猪和大鼠血清白蛋白的¹H NMR谱。通过序列比较，对牛（天冬氨酸-苏氨酸-组氨酸）、人（天冬氨酸-丙氨酸-组氨酸）、猪（天冬氨酸-苏氨酸-酪氨酸）和大鼠（谷氨酸-丙氨酸-组氨酸）血清白蛋白的N端残基，以及牛血清白蛋白的Thr189、Tyr155和His59/377进行了具体归属。7. 这些NMR数据表明，牛血清白蛋白的某些局部区域在溶液中高度可移动但具有结构，并证明一维和二维NMR方法的应用将允许更详细地研究例如pH、药物和金属结合诱导的血清白蛋白结构转变。
