Centro de Ciências Naturais e Humanas, Universidade Federal do ABC Santo André, Brazil.
Front Chem. 2016 Mar 31;4:13. doi: 10.3389/fchem.2016.00013. eCollection 2016.
The present study aimed to investigate the influence of albumin structure and gold speciation on the synthesis of gold nanoparticles (GNPs). The strategy of synthesis was the addition of HAuCl4 solutions at different pH values (3-12) to solutions of human and bovine serum albumins (HSA and BSA) at the same corresponding pH values. Different pH values influence the GNP synthesis due to gold speciation. Besides the inherent effect of pH on the native structure of albumins, the use N-ethylmaleimide (NEM)-treated and heat-denaturated forms of HSA and BSA provided additional insights about the influence of protein structure, net charge, and thiol group approachability on the GNP synthesis. NEM treatment, heating, and the extreme values of pH promoted loss of the native albumin structure. The formation of GNPs indicated by the appearance of surface plasmon resonance (SPR) bands became detectable from 15 days of the synthesis processes that were carried out with native, NEM-treated and heat-denaturated forms of HSA and BSA, exclusively at pH 6 and 7. After 2 months of incubation, SPR band was also detected for all synthesis carried out at pH 8.0. The mean values of the hydrodynamic radius (RH) were 24 and 34 nm for GNPs synthesized with native HSA and BSA, respectively. X-ray diffraction (XRD) revealed crystallites of 13 nm. RH, XRD, and zeta potential values were consistent with GNP capping by the albumins. However, the GNPs produced with NEM-treated and heat-denaturated albumins exhibited loss of protein capping by lowering the ionic strength. This result suggests a significant contribution of non-electrostatic interactions of albumins with the GNP surface, in these conditions. The denaturation of proteins exposes hydrophobic groups to the solvent, and these groups could interact with the gold surface. In these conditions, the thiol blockage or oxidation, the latter probably favored upon heating, impaired the formation of a stable capping by thiol coordination with the gold surface. Therefore, the cysteine side chain of albumins is important for the colloidal stabilization of GNPs rather than as the reducing agent for the synthesis. Despite the presence of more reactive gold species at more acidic pH values, i.e., below 6.0, in these conditions the loss of native albumin structure impaired GNP synthesis. Alkaline pH values (9-12) combined the unfavorable conditions of denaturated protein structure with less reactive gold species. Therefore, an optimal condition for the synthesis of GNPs using serum albumins involves more reactive gold salt species combined with a reducing and negatively charged form of the protein, all favored at pH 6-7.
本研究旨在探讨白蛋白结构和金的形态对金纳米粒子（GNPs）合成的影响。合成策略是在相同的相应 pH 值下，将 HAuCl4 溶液添加到人血清白蛋白（HSA）和牛血清白蛋白（BSA）溶液中。不同的 pH 值会影响 GNP 的合成，这是由于金的形态不同。除了 pH 值对白蛋白固有结构的内在影响外，使用 N-乙基马来酰亚胺（NEM）处理和热变性的 HSA 和 BSA 形式还提供了有关蛋白质结构、净电荷和巯基接近度对 GNP 合成影响的更多信息。NEM 处理、加热和 pH 值的极值促进了天然白蛋白结构的丧失。通过出现表面等离子体共振（SPR）带表明形成了 GNPs，从用天然、NEM 处理和热变性的 HSA 和 BSA 进行合成的 15 天开始可以检测到 SPR 带，仅在 pH 值为 6 和 7 时才可以检测到。孵育 2 个月后，所有在 pH 值为 8.0 下进行的合成也检测到了 SPR 带。用天然 HSA 和 BSA 合成的 GNPs 的平均水动力半径（RH）分别为 24nm 和 34nm。X 射线衍射（XRD）显示出 13nm 的结晶。RH、XRD 和 ζ 电位值与白蛋白包被的 GNPs 一致。然而，用 NEM 处理和热变性的白蛋白合成的 GNPs 由于离子强度降低而失去了蛋白包被。结果表明，在这些条件下，白蛋白与 GNP 表面的非静电相互作用有显著贡献。蛋白质的变性使疏水性基团暴露于溶剂中，这些基团可以与金表面相互作用。在这些条件下，巯基的阻断或氧化（可能在加热时更有利于后者）会损害通过巯基与金表面的配位形成稳定的包被。因此，白蛋白的半胱氨酸侧链对于 GNPs 的胶体稳定性很重要，而不是作为合成的还原剂。尽管在更酸性的 pH 值（<6.0）下存在更具反应性的金物种，但在这些条件下，天然白蛋白结构的丧失会损害 GNP 的合成。碱性 pH 值（9-12）结合了变性蛋白结构的不利条件和较少反应性的金物种。因此，使用血清白蛋白合成 GNPs 的最佳条件是结合更具反应性的金盐物种和具有还原和负电荷的蛋白质形式，所有这些都在 pH 值为 6-7 时得到促进。
