School of Agriculture and Food Sciences, The University of Queensland, Brisbane QLD 4072, Australia.
J Dairy Res. 2013 Nov;80(4):383-9. doi: 10.1017/S0022029913000332. Epub 2013 Jul 19.
Prevention of the heat-induced aggregation of β-lactoglobulin (β-Lg) would improve the heat stability of whey proteins. The effects of lipoic acid (LA, or thioctic acid), in both its oxidised and reduced form (dihydrolipoic acid, DHLA), on heat-induced unfolding and aggregation of β-Lg were investigated. LA/DHLA was added to native β-Lg and the mixture was heated at 70, 75, 80 or 85 °C for up to 30 min at pH 6·8. The samples were analysed by Polyacrylamide Gel Electrophoresis (PAGE) and Size-exclusion HPLC (SE-HPLC). LA was not as effective as DHLA in reducing the formation of aggregates of heated β-Lg. Heating β-Lg with DHLA resulted in formation of more β-Lg monomers (due to dissociation of native dimers) and significantly less β-Lg aggregates, compared with heating β-Lg alone. The aggregates formed in the presence of DHLA were both covalently linked, via disulphide bonds, and non-covalently (hydrophobically) linked, but the amount of covalently linked aggregates was much less than when β-Lg was heated alone. The results suggest that DHLA was able to partially trap the reactive β-Lg monomer containing a free sulphydryl (-SH) group, by forming a 'modified monomer', and to prevent some sulphydryl-sulphydryl and sulphydryl-disulphide interactions that lead to the formation of covalently linked protein aggregates. The effects of DHLA were similar to those of N-ethylmaleimide (NEM) and dithio(bis)-p-nitrobenzoate (DTNB). However, the advantage of using DHLA over NEM and DTNB to lessen aggregation of β-Lg is that it is a food-grade compound which occurs naturally in milk.
预防β-乳球蛋白（β-Lg）的热诱导聚集可以提高乳清蛋白的热稳定性。研究了两种形式的硫辛酸（LA，或硫辛酸），即氧化形式（二氢硫辛酸，DHLA）和还原形式，对β-Lg 的热诱导变性和聚集的影响。将 LA/DHLA 添加到天然的β-Lg 中，将混合物在 pH6.8 下在 70、75、80 或 85°C 加热长达 30 分钟。通过聚丙烯酰胺凝胶电泳（PAGE）和尺寸排阻高效液相色谱（SE-HPLC）分析样品。LA 不如 DHLA 有效，无法减少加热的β-Lg 聚集物的形成。与单独加热β-Lg 相比，用 DHLA 加热β-Lg 会导致更多的β-Lg 单体（由于天然二聚体的解离）形成，并且显著减少β-Lg 聚集物的形成。在 DHLA 存在下形成的聚集物通过二硫键共价连接，并且通过疏水相互作用非共价连接，但是共价连接的聚集物的量比单独加热β-Lg 时少得多。结果表明，DHLA 能够通过形成“修饰单体”部分捕获含有游离巯基（-SH）基团的反应性β-Lg 单体，并防止一些导致形成共价连接的蛋白质聚集物的巯基-巯基和巯基-二硫键相互作用。DHLA 的作用类似于 N-乙基马来酰亚胺（NEM）和二硫代双（对硝基苯甲酸酯）（DTNB）。然而，与 NEM 和 DTNB 相比，使用 DHLA 来减少β-Lg 的聚集的优点在于它是一种食品级化合物，天然存在于牛奶中。
