Parks J S, Cistola D P, Small D M, Hamilton J A
J Biol Chem. 1983 Aug 10;258(15):9262-9.
The interactions of the carboxyl group of oleic acid with bovine serum albumin (BSA) were studied by 13C NMR spectroscopy at 50.3 MHz using 90% isotopically substituted [1-13C]oleic acid. 13C NMR spectra were obtained as a function of the mole ratio of oleic acid to BSA (from 0.5-10.0) and, for selected mole ratios, as a function of pH (between pH 3.0 and 10.6) and temperature (between 15 and 55 degrees C and thermally denatured at 95 degrees C). Except for spectra of highly acidic (pH less than or equal to 3.9) and denatured samples, spectra of oleic acid/BSA complexes showed multiple narrow resonances from the oleic acid carboxyl carbon in a region (179-184 ppm) downfield from protein carbonyl and carboxyl carbon resonances. At low oleic acid/BSA ratios (0.5 and 1.0), at least two oleic acid carboxyl carbon peaks were observed; at high ratios (greater than or equal to 3.0), at least four peaks were present. The intensities of individual peaks, but not their chemical shifts, varied with the oleic acid/BSA ratio. The chemical shift of individual oleic acid peaks was invariant between pH 6.0 and 10.6; below pH 6.0, one of the oleic acid resonances exhibited an NMR titration curve with an apparent pKa of approximately 4. Thus, BSA binding sites for oleic acid are heterogeneous as monitored by the magnetic microenvironment of the oleic acid carboxyl carbon. The number of different oleic acid environments and the relative population of oleic acid molecules in these environments is dependent on the mole ratio of oleic acid/BSA. Our results suggested that the anionic form of oleic acid is bound to BSA at physiological pH and that the multiplicity of NMR peaks for [1-13C]oleic acid resulted from, at least in part, different electrostatic and hydrogen bonding interactions between the oleic acid carboxyl group and specific amino acid residues of BSA.
使用90%同位素取代的[1-¹³C]油酸，通过50.3 MHz的¹³C核磁共振光谱研究了油酸羧基与牛血清白蛋白（BSA）的相互作用。¹³C核磁共振光谱是作为油酸与BSA摩尔比（从0.5至10.0）的函数获得的，并且对于选定的摩尔比，是作为pH值（在pH 3.0至10.6之间）和温度（在15至55摄氏度之间，并在95摄氏度下热变性）的函数获得的。除了高酸性（pH小于或等于3.9）和变性样品的光谱外，油酸/BSA复合物的光谱在蛋白质羰基和羧基碳共振的低场区域（179 - 184 ppm）显示出来自油酸羧基碳的多个窄共振峰。在低油酸/BSA比率（0.5和1.0）下，观察到至少两个油酸羧基碳峰；在高比率（大于或等于3.0）下，存在至少四个峰。各个峰的强度而非其化学位移随油酸/BSA比率而变化。各个油酸峰的化学位移在pH 6.0至10.6之间不变；在pH 6.0以下，其中一个油酸共振峰呈现出具有约4的表观pKa的核磁共振滴定曲线。因此，通过油酸羧基碳的磁性微环境监测，BSA上油酸的结合位点是异质的。不同油酸环境的数量以及这些环境中油酸分子的相对数量取决于油酸/BSA的摩尔比。我们的结果表明，在生理pH下，油酸的阴离子形式与BSA结合，并且[1-¹³C]油酸核磁共振峰的多重性至少部分是由于油酸羧基与BSA特定氨基酸残基之间不同的静电和氢键相互作用所致。
