Department of Chemistry, Sapienza University of Rome, P. le A. Moro 5, 00185 Rome, Italy.
Department of Chemistry, Sapienza University of Rome, P. le A. Moro 5, 00185 Rome, Italy.
Colloids Surf B Biointerfaces. 2018 Aug 1;168:109-116. doi: 10.1016/j.colsurfb.2018.03.038. Epub 2018 Mar 27.
The protein Human Serum Albumin (HSA) is known to undergo conformational transitions towards partially unfolded forms triggered by acidification below pH 4.5. The extent of Fatty Acids (FA) binding has been thought to have an impact on the conformational equilibrium between the native and acid forms and to be a possible explanation for the observation of more than one band in early electrophoretic migration experiments at pH 4. We compared the acid-induced unfolding processes of commercial FA-free HSA, commercial "fatted" HSA and FA-HSA complexes, prepared at FA:HSA molar ratios between 1 and 6 by simple mixing and equilibration. We used a method for continuous acidification based on the hydrolysis of glucono-δ-lactone from pH 7 to pH 2.5, and followed the average protein changes by the blue shift of the intrinsic fluorescence emission and by performing a small angle X-ray scattering analysis on selected samples. The method also allowed for continuous monitoring of the increase of turbidity and laser light scattering of the protein samples related to the release of the insoluble ligands with acidification. Our results showed that the presence of FA interacting with albumin, an aspect often neglected in biophysical studies, affects the conformational response of the protein to acidification, and slightly shifts the loss of the native shape from pH 4.2 to pH 3.6. This effect increased with the FA:HSA molar ratio so that with three molar equivalents a saturation was reached, in agreement with the number of high-affinity binding sites reported for the FA. These findings confirm that a non-uniform level of ligand binding in an albumin sample can be an explanation for the early-observed conformational heterogeneity at pH 4.
人血清白蛋白（HSA）蛋白已知会发生构象转变，向部分展开形式转变，这是由 pH 值低于 4.5 的酸化触发的。脂肪酸（FA）结合的程度被认为对 native 和 acid 形式之间的构象平衡有影响，并可能是在 pH 值为 4 时观察到多个条带的早期电泳迁移实验的一个可能解释。我们比较了商业 FA 游离 HSA、商业“加脂”HSA 和 FA-HSA 复合物在 FA:HSA 摩尔比为 1 到 6 之间通过简单混合和平衡制备时的酸诱导展开过程。我们使用了一种基于葡萄糖酸-δ-内酯从 pH 7 水解到 pH 2.5 的连续酸化方法，并通过内源荧光发射的蓝移和对选定样品进行小角度 X 射线散射分析来跟踪平均蛋白质变化。该方法还允许连续监测与酸化相关的不溶性配体释放导致的蛋白质样品浊度和激光光散射的增加。我们的结果表明，FA 与白蛋白相互作用的存在，这是生物物理研究中经常被忽视的一个方面，会影响蛋白质对酸化的构象响应，并将 native 形状的丧失略微从 pH 值 4.2 转移到 pH 值 3.6。这种效应随着 FA:HSA 摩尔比的增加而增加，因此当达到三个摩尔当量时达到饱和，这与 FA 报道的高亲和力结合位点的数量一致。这些发现证实，在白蛋白样品中配体结合的不均匀水平可以解释在 pH 值 4 时观察到的早期构象异质性。
