Characterization of the covalent binding of allyl isothiocyanate to β-lactoglobulin by fluorescence quenching, equilibrium measurement, and mass spectrometry
Characterization of the covalent binding of allyl isothiocyanate to β-lactoglobulin by fluorescence quenching, equilibrium measurement, and mass spectrometry
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DOI：
10.1080/07391102.2013.809605
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发表时间：
2014-07-03
影响因子：
4.4
通讯作者：
Schwarz, Karin
中科院分区：
文献类型：
--
作者：
Keppler, Julia Katharina;Koudelka, Tomas;Schwarz, Karin
小分子化合物通过疏水相互作用或氢键与食物蛋白质（如牛奶蛋白）的可逆结合是一个深入研究的课题。相反，共价相互作用没有得到很好的表征。在这里，我们报告了一种罕见的形式的正协同线性结合的烯丙基异硫氰酸酯与β-乳球蛋白，导致蛋白质的二硫键的裂解。我们比较了三种方法（即荧光猝灭，平衡透析，和顶空-水平衡）来表征结合动力学，并通过质谱研究了分子结合。所使用的方法被认为是相当的和可重复的存在下，高和低的配体浓度的荧光猝灭和平衡为基础的方法分别。
Reversible binding of small compounds through hydrophobic interactions or hydrogen bonding to food proteins (e.g. milk proteins) is a thoroughly researched topic. In contrast, covalent interactions are not well characterized. Here, we report a rare form of positive-cooperativity-linear binding of allyl isothiocyanate with beta-lactoglobulin, resulting in the cleavage of a disulfide bond of the protein. We compared three methods (i.e. fluorescence quenching, equilibrium dialysis, and headspace-water equilibrium) to characterize the binding kinetics and investigated the molecular binding by mass spectrometry. The methodologies used were found to be comparable and reproducible in the presence of high and low ligand concentrations for fluorescence quenching and equilibrium-based methods respectively.