Martins P A T, Gomes F, Vaz W L C, Moreno M J
Departamento de Química da FCTUC, Rua Larga, 3004-535 Coimbra, Portugal.
Biochim Biophys Acta. 2008 May;1778(5):1308-15. doi: 10.1016/j.bbamem.2008.02.011. Epub 2008 Mar 4.
The bovine milk lipocalin, beta-Lactoglobulin (beta-LG), has been associated with the binding and transport of small hydrophobic and amphiphilic compounds, whereby it is proposed to increase their bioavailability. We have studied the binding of the fluorescent phospholipid-derivative, NBD-didecanoylphosphatidylethanolamine (NBD-diC10PE) to beta-LG by following the increase in amphiphile fluorescence upon binding to the protein using established methods. The equilibrium association constant, KB, was (1.2+/-0.2)x10(6) M(-1) at 25 degrees C, pH 7.4 and I=0.15 M. Dependence of KB on pH and on the monomer-dimer equilibrium of beta-LG gave insight on the nature of the binding site which is proposed to be the hydrophobic calyx formed by the beta-barrel in the protein. The monomer-dimer equilibrium of beta-LG was re-assessed using fluorescence anisotropy of Tryptophan. The equilibrium constant for dimerization, KD, was (7.0+/-1.5)x10(5) M(-1) at 25 degrees C, pH 7.4, and 0.15 M ionic strength. The exchange of NBD-diC10PE between beta-LG and POPC lipid bilayers was followed by the change in NBD fluorescence. beta-LG was shown to be a catalyst of phospholipid exchange between lipid bilayers, the mechanism possibly involving adsorption of the protein at the bilayer surface.
牛乳脂钙蛋白，即β-乳球蛋白（β-LG），与小的疏水性及两亲性化合物的结合和运输有关，因此有人提出它能提高这些化合物的生物利用度。我们通过使用已建立的方法，跟踪两亲性荧光在与蛋白质结合时的增加情况，研究了荧光磷脂衍生物NBD-二癸酰磷脂酰乙醇胺（NBD-diC10PE）与β-LG的结合。在25℃、pH 7.4和I = 0.15 M条件下，平衡缔合常数KB为(1.2±0.2)×10⁶ M⁻¹。KB对pH和β-LG单体-二聚体平衡的依赖性揭示了结合位点的性质，该位点被认为是由蛋白质中的β-桶形成的疏水萼。利用色氨酸的荧光各向异性重新评估了β-LG的单体-二聚体平衡。在25℃、pH 7.4和离子强度为0.15 M的条件下，二聚化平衡常数KD为(7.0±1.5)×10⁵ M⁻¹。通过NBD荧光的变化跟踪了NBD-diC10PE在β-LG和POPC脂质双层之间的交换。结果表明β-LG是脂质双层之间磷脂交换的催化剂，其机制可能涉及蛋白质在双层表面的吸附。
