Dave Anant C, Loveday Simon M, Anema Skelte G, Jameson Geoffrey B, Singh Harjinder
Riddet Institute, Massey University , Private Bag 11 222, Palmerston North, New Zealand.
Biomacromolecules. 2014 Jan 13;15(1):95-103. doi: 10.1021/bm401315s. Epub 2013 Dec 23.
β-Lactoglobulin (β-lg) forms fibrils when heated at 80 °C, pH 2, and low ionic strength (<0.015 mM). When formed at protein concentrations <3%, these fibrils are made up of peptides produced from the acid hydrolysis of the β-lg monomer. The present study investigated the effects of the polyhydroxy alcohols (polyols) glycerol and sorbitol (0-50% w/v) on β-lg self-assembly at pH 2. Glycerol and sorbitol stabilize native protein structure and modulate protein functionality by preferential exclusion. In our study, both polyols decreased the rate of β-lg self-assembly but had no effect on the morphology of fibrils. The mechanism of these effects was studied using circular dichroism spectroscopy and SDS-PAGE. Sorbitol inhibited self-assembly by stabilizing β-lg against unfolding and hydrolysis, resulting in fewer fibrillogenic species, whereas glycerol inhibited nucleation without inhibiting hydrolysis. Both polyols increased the viscosity of the solutions, but viscosity appeared to have little effect on fibril assembly, and we believe that self-assembly was not diffusion-limited under these conditions. This is in agreement with previous reports for other proteins assembling under different conditions. The phenomenon of peptide self-assembly can be decoupled from protein hydrolysis using glycerol.
β-乳球蛋白（β-lg）在80℃、pH 2和低离子强度（<0.015 mM）条件下加热时会形成纤维。当在蛋白质浓度<3%时形成这些纤维时，它们由β-lg单体酸水解产生的肽组成。本研究考察了多元醇甘油和山梨醇（0 - 50% w/v）在pH 2条件下对β-lg自组装的影响。甘油和山梨醇通过优先排阻作用稳定天然蛋白质结构并调节蛋白质功能。在我们的研究中，两种多元醇都降低了β-lg自组装的速率，但对纤维的形态没有影响。使用圆二色光谱和SDS - PAGE研究了这些作用的机制。山梨醇通过稳定β-lg防止其展开和水解来抑制自组装，从而产生较少的成纤维物种，而甘油抑制成核但不抑制水解。两种多元醇都增加了溶液的粘度，但粘度似乎对纤维组装影响不大，并且我们认为在这些条件下自组装不是扩散限制的。这与之前关于其他蛋白质在不同条件下组装的报道一致。使用甘油可以使肽自组装现象与蛋白质水解解耦。
