Wang Q, Allen J C, Swaisgood H E
Department of Food Science, North Carolina State University, Raleigh 27695-7624, USA.
J Dairy Sci. 1997 Jun;80(6):1047-53. doi: 10.3168/jds.S0022-0302(97)76029-6.
Binding of the retinoids, all-trans-retinol, all-trans-retinal, all-trans-retinyl acetate, and all-trans-retinoic acid, to beta-lactoglobulin (LG) (96% purity) that had been prepared by bioselective adsorption on N-retinyl-Celite was determined from changes in the fluorescence quenching (332 nm) of the protein tryptophanyl residues. High affinity binding of all of these compounds occurred at pH 7.0, and the apparent dissociation constant ranged from 1.7 to 3.6 x 10(-8) M. Furthermore, a stoichiometry of 1.0 mol.mol-1 of protein was obtained for each case, indicating that all of the sites in the protein preparation were available. When beta-LG in whey protein isolate (57.4% beta-LG) was studied, a stoichiometry of 0.65 to 0.82 mol.mol-1 of protein was obtained, indicating that a large number of the sites already had bound lipid or that the protein had been denatured. As the pH was lowered toward 5.15, the affinity decreased about fourfold, but the stoichiometry of binding was unchanged. Far UV circular dichroism spectra indicated that the secondary structure of the protein was not significantly affected by ligand binding; however, the near UV spectra were changed, indicating that the flexibility of tryptophanyl residues decreased. The latter effect is consistent with the change in fluorescence quenching and suggests that a tryptophan is in the binding site.
通过蛋白质色氨酸残基荧光猝灭（332nm）的变化，测定了视黄醇类物质，即全反式视黄醇、全反式视黄醛、全反式乙酸视黄酯和全反式视黄酸，与通过生物选择性吸附在N-视黄基-硅藻土上制备的β-乳球蛋白（LG）（纯度96%）的结合情况。所有这些化合物在pH 7.0时均发生高亲和力结合，表观解离常数范围为1.7至3.6×10⁻⁸M。此外，每种情况下蛋白质的化学计量比均为1.0 mol·mol⁻¹，表明蛋白质制剂中的所有位点均可用。当研究乳清蛋白分离物中的β-LG（57.4%β-LG）时，获得的蛋白质化学计量比为0.65至0.82 mol·mol⁻¹，表明大量位点已结合脂质或蛋白质已变性。随着pH降至5.15，亲和力降低约四倍，但结合的化学计量比不变。远紫外圆二色光谱表明，配体结合对蛋白质的二级结构没有显著影响；然而，近紫外光谱发生了变化，表明色氨酸残基的灵活性降低。后一种效应与荧光猝灭的变化一致，表明色氨酸位于结合位点。
