Cistola D P, Small D M, Hamilton J A
J Biol Chem. 1987 Aug 15;262(23):10980-5.
13C NMR chemical shift results as a function of pH for a series of carboxyl 13C-enriched saturated fatty acids (8-18 carbons) bound to bovine serum albumin (BSA) are presented. For octanoic acid bound to BSA (6:1, mol/mol), the chemical shift of the only FA carboxyl resonance (designated as peak c), plotted as a function of pH, exhibited a complete sigmoidal titration curve that deviated in shape from a corresponding theoretical Henderson-Hasselbach curve. However, FA carboxyl chemical shift plotted as a function of added HCl yielded a linear titration curve analogous to those obtained for protein-free monomeric fatty acid (FA) in water. The apparent pK of BSA-bound octanoic acid was 4.3 +/- 0.2. However, the intrinsic pK (corrected for electrostatic effects resulting from the net positive charge on BSA) was approximately 4.8, a value identical to that obtained for monomeric octanoic acid in water in the absence of protein. For long-chain FA (greater than or equal to 12 carbons) bound to BSA (6:1, mol/mol), chemical shift titration curves for peak c were similar to those obtained for octanoic acid/BSA. However, the four additional FA carboxyl resonances observed (designated as peaks a, b, b', and d) exhibited no change in chemical shift between pH 8 and 3. For C14.0 X BSA complexes (3:1 and 6:1, mol/mol) peaks b' and a exhibited chemical shift changes between pH 8.8 and 11.5 concomitant with chemical shift changes in the epsilon-carbon (lysine) resonance. In contrast, peaks c and d exhibited no change and peak b only a slight change in chemical shift over the same pH range. We conclude: the carboxyl groups of bound FA represented by peaks a, b, b', and d were involved in ion pair electrostatic interactions with positively charged amino acyl residues on BSA; the carboxyl groups of bound FA represented by peak c were not involved in electrostatic interactions with BSA; the similarity of the titration curves of peak c for BSA-bound octanoic acid and long-chain FA suggested that short-chain and long-chain FA represented by peak c were bound to the same binding site(s) on BSA; bound FA represented by peaks b' and a (but not d or b) were directly adjacent to BSA lysine residues. We present a model which correlates NMR peaks b, b', and d with the putative locations of three individual high-affinity binding sites in a three-dimensional model of BSA.
本文给出了一系列羧基富含(^{13}C)的饱和脂肪酸（8 - 18个碳）与牛血清白蛋白（BSA）结合时，(^{13}C) NMR化学位移随pH的变化结果。对于与BSA结合的辛酸（6:1，摩尔/摩尔），唯一的脂肪酸羧基共振峰（标记为峰c）的化学位移随pH绘制，呈现出完整的S形滴定曲线，其形状与相应的理论亨德森 - 哈塞尔巴赫曲线不同。然而，脂肪酸羧基化学位移随添加HCl绘制得到的线性滴定曲线类似于在水中无蛋白的单体脂肪酸（FA）所得到的曲线。与BSA结合的辛酸的表观(pK)为4.3 ± 0.2。然而，内在(pK)（校正了由于BSA上的净正电荷产生的静电效应）约为4.8，这一数值与在无蛋白的水中单体辛酸所得到的数值相同。对于与BSA结合的长链脂肪酸（大于或等于12个碳，6:1，摩尔/摩尔），峰c的化学位移滴定曲线与辛酸/BSA的相似。然而，观察到的另外四个脂肪酸羧基共振峰（标记为峰a、b、b'和d）在pH 8至3之间化学位移没有变化。对于(C14.0)与BSA的复合物（3:1和6:1，摩尔/摩尔），峰b'和a在pH 8.8至11.5之间化学位移发生变化，同时ε - 碳（赖氨酸）共振化学位移也发生变化。相反，在相同pH范围内，峰c和d化学位移无变化，峰b仅有轻微变化。我们得出结论：由峰a、b、b'和d代表的结合脂肪酸的羧基参与了与BSA上带正电荷的氨基酰基残基的离子对静电相互作用；由峰c代表的结合脂肪酸的羧基不参与与BSA的静电相互作用；与BSA结合的辛酸和长链脂肪酸峰c的滴定曲线相似，表明由峰c代表的短链和长链脂肪酸与BSA上的相同结合位点结合；由峰b'和a（而非d或b）代表的结合脂肪酸直接与BSA赖氨酸残基相邻。我们提出了一个模型，将NMR峰b、b'和d与BSA三维模型中三个单独的高亲和力结合位点的假定位置相关联。
