Control of α-Lactalbumin Aggregation by Modulation of Temperature and Concentration of Calcium and Cysteine,Journal of Agricultural and Food Chemistry

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Control of α-Lactalbumin Aggregation by Modulation of Temperature and Concentration of Calcium and Cysteine
Journal of Agricultural and Food Chemistry ( IF 5.7 ) Pub Date : 2018-06-19 00:00:00 , DOI: 10.1021/acs.jafc.8b01172
Line R. Nielsen _{1,

2} , Søren B. Nielsen ₁ , Zichen Zhao ₂ , Karsten Olsen ₂ , Jacob H. Nielsen ₁ , Marianne N. Lund _{2,

3}

Affiliation

The effect of free cysteine (in different concentrations) on the thermal aggregation of calcium-saturated (Ca-sat) and -depleted (Ca-dep) α-lactalbumin (α-LA) was investigated at 25, 50, and 70 °C. The temperatures chosen were below the denaturation temperature (T_d) of Ca-dep and Ca-sat α-LA (25 °C), above the T_d of Ca-dep α-LA and below that of Ca-sat α-LA (50 °C), and above the T_d of Ca-sat α-LA (70 °C). Size-exclusion chromatography coupled to multiangle light scattering showed that no aggregation or only minor aggregation was obtained at the investigated temperatures for both Ca-dep and Ca-sat α-LA even at extended holding times. Aggregates of Ca-sat α-LA were larger than those developed for Ca-dep α-LA. The addition of cysteine, a low-molecular-mass free thiol, resulted in increased aggregation of both Ca-sat and Ca-dep α-LA. Comparisons of SDS-PAGE run under reducing and nonreducing conditions showed that the formed cross-links were primarily disulfide bonds, but Western blots also showed small contributions from dityrosine cross-link formation. The aggregation kinetics related to monomer loss during heat treatment were determined by RP-UPLC and showed that the addition of cysteine increased the rate of aggregation. The activation energies for Ca-dep α-LA with 0.35 and 0.7 mM cysteine were found to be 59 ± 1 and 46 ± 4 kJ/mol, respectively, which showed that less energy was needed for the enhanced thermal aggregation of α-LA when the cysteine concentration was increased. This study showed that it was possible to control the aggregation size of α-LA by manipulating the incubation temperature and the cysteine concentration.

中文翻译：

通过调节温度以及钙和半胱氨酸的浓度来控制α-乳清蛋白聚集

在25、50和70°C下研究了游离半胱氨酸（不同浓度）对饱和钙（Ca-sat）和贫钙（Ca-dep）α-乳清蛋白（α-LA）的热聚集的影响。选择的温度为低于变性温度（Ť _d的Ca-DEP和Ca-SATα-LA（25℃），以上所述的）Ť _d的Ca-DEPα-LA的和低于的钙饱和α-LA （50°C），且高于T _dCa-satα-LA（70°C）。与多角度光散射耦合的尺寸排阻色谱法表明，即使在延长的保温时间下，对于所研究的Ca-dep和Ca-satα-LA而言，均未获得聚集或仅发生少量聚集。Ca-satα-LA的聚集体大于为Ca-depα-LA形成的聚集体。半胱氨酸（一种低分子质量的游离硫醇）的添加导致Ca-sat和Ca-depα-LA的聚集增加。在还原和非还原条件下进行SDS-PAGE的比较表明，形成的交联主要是二硫键，但Western印迹也显示二氢酪氨酸交联形成的贡献很小。通过RP-UPLC测定与热处理过程中单体损失有关的聚集动力学，结果表明添加半胱氨酸可提高聚集速率。发现具有0.35和0.7 mM半胱氨酸的Ca-depα-LA的活化能分别为59±1和46±4 kJ / mol，这表明当α-LA增强热聚集时所需的能量更少。半胱氨酸浓度增加。这项研究表明，可以通过控制孵育温度和半胱氨酸浓度来控制α-LA的聚集大小。

更新日期：2018-06-19

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