Muresan S, van der Bent A, de Wolf F A
Agrotechnological Research Institute ATO, Wageningen University and Research Center, P.O. Box 17, 6700 AA Wageningen, The Netherlands.
J Agric Food Chem. 2001 May;49(5):2609-18. doi: 10.1021/jf0012188.
Although a thorough characterization of binding parameters is essential for application of beta-lactoglobulin as a carrier for a variety of small hydrophobic ligands, the binding parameters derived in various studies using various techniques are inconsistent. The bindings of several small ligands as detected by fluorometry and equilibrium dialysis were compared. Fluorescence spectroscopy showed that beta-ionone, retinol, and fatty acid lactones all bound in the vicinity of a tryptophan residue. Retinol and fatty acid lactone competed for the same binding site. Exclusively for ligands that quench the beta-lactoglobulin fluorescence through a resonance energy transfer mechanism, fluorometry yielded a systematically higher binding affinity than equilibrium dialysis. The binding overestimation in fluorometric measurements can be explained by oligomer formation of protein, together with an underestimation of the limiting quenching level at saturating ligand concentrations due to the use of a limited set of data points.
尽管全面表征结合参数对于将β-乳球蛋白用作多种小疏水配体的载体至关重要，但使用各种技术在不同研究中得出的结合参数并不一致。比较了通过荧光测定法和平衡透析检测到的几种小配体的结合情况。荧光光谱表明，β-紫罗兰酮、视黄醇和脂肪酸内酯均在色氨酸残基附近结合。视黄醇和脂肪酸内酯竞争相同的结合位点。仅对于通过共振能量转移机制淬灭β-乳球蛋白荧光的配体，荧光测定法产生的结合亲和力系统地高于平衡透析法。荧光测量中的结合高估可以通过蛋白质的寡聚体形成来解释，同时由于使用有限的数据点集，在饱和配体浓度下对极限淬灭水平的低估。
