Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC 3800, Australia.
Langmuir. 2012 Feb 7;28(5):2357-67. doi: 10.1021/la203281c. Epub 2012 Jan 18.
The conformation and structural dimensions of α-lactalbumin (α-La) both in solution and adsorbed at oil-water interfaces of emulsions were investigated using synchrotron radiation circular dichroism (SRCD) spectroscopy, front-face tryptophan fluorescence (FFTF) spectroscopy, and dual polarization interferometry (DPI). The near-UV SRCD and the FFTF results demonstrated that the hydrophobic environment of the aromatic residues located in the hydrophobic core of native α-La was significantly altered upon adsorption, indicating the unfolding of the hydrophobic core of α-La upon adsorption. The far-UV SRCD results showed that adsorption of α-La at oil-water interfaces created a new non-native secondary structure that was more stable to thermally induced conformational changes. Specifically, the α-helical conformation increased from 29.9% in solution to 45.8% at the tricaprylin-water interface and to 58.5% at the hexadecane-water interface. However, the β-sheet structure decreased from 18.0% in solution to less than 10% at both oil-water interfaces. The DPI study showed that adsorption of α-La to a hydrophobic C18-water surface caused a change in the dimensions of α-La from the native globule-like shape (2.5-3.7 nm) to a compact/dense layer approximately 1.1 nm thick. Analysis of the colloidal stability of α-La stabilized emulsions showed that these emulsions were physically stable against droplet flocculation at elevated temperatures both in the absence and in the presence of 120 mM NaCl. In the absence of salt, the thermal stability of emulsions was due to the strong electrostatic repulsion provided by the adsorbed α-La layer, which was formed after the adsorption and structural rearrangement. In the presence of salt, although the electrostatic repulsion was reduced via electrostatic screening, heating did not induce strong and permanent droplet flocculation. The thermal stability of α-La stabilized emulsions in the presence of salt is a combined effect of the electrostatic repulsion and the lack of covalent disulfide interchange reactions. This study reports new information on the secondary and tertiary structural changes of α-La upon adsorption to oil-water interfaces. It also presents new results on the physical stability of α-La stabilized emulsions during heating and at moderate ionic strength (120 mM NaCl). The results broaden our understanding of the factors controlling protein structural change at emulsion interfaces and how this affects emulsion stability.
使用同步辐射圆二色性（SRCD）光谱、正面色氨酸荧光（FFTF）光谱和双偏振干涉测量（DPI）研究了α-乳白蛋白（α-La）在溶液中和吸附在乳液油水界面时的构象和结构尺寸。近紫外 SRCD 和 FFTF 结果表明，吸附时位于天然α-La 疏水区的芳香族残基的疏水环境发生了显著变化，表明α-La 疏水区在吸附时展开。远紫外 SRCD 结果表明，α-La 在油水界面的吸附形成了一种新的非天然二级结构，对热诱导构象变化更稳定。具体而言，α-螺旋构象从溶液中的 29.9%增加到三辛酸酯-水界面的 45.8%和十六烷-水界面的 58.5%。然而，β-折叠结构从溶液中的 18.0%减少到两个油水界面的不到 10%。DPI 研究表明，α-La 吸附到疏水 C18-水表面会导致α-La 的尺寸从天然球状形状（2.5-3.7nm）变为约 1.1nm 厚的紧凑/密集层。对α-La 稳定乳液的胶体稳定性分析表明，这些乳液在高温下对液滴聚结是物理稳定的，无论是在没有盐的情况下还是在存在 120mM NaCl 的情况下都是如此。在没有盐的情况下，乳液的热稳定性归因于吸附α-La 层提供的强静电排斥，该层在吸附和结构重排后形成。在存在盐的情况下，尽管静电排斥通过静电屏蔽而降低，但加热不会引起强烈和永久的液滴聚结。在存在盐的情况下，α-La 稳定乳液的热稳定性是静电排斥和缺乏共价二硫键交换反应的综合效应。本研究报告了α-La 吸附到油水界面时二级和三级结构变化的新信息。它还提供了有关在加热和中等离子强度（120mM NaCl）下α-La 稳定乳液的物理稳定性的新结果。研究结果拓宽了我们对控制乳液界面上蛋白质结构变化的因素以及这如何影响乳液稳定性的理解。
