Putilina T, Sittenfeld D, Chader G J, Wiggert B
Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892.
Biochemistry. 1993 Apr 13;32(14):3797-803. doi: 10.1021/bi00065a036.
Interphotoreceptor retinoid-binding protein (IRBP) is a 140-kDa glycolipoprotein which constitutes about 70% of the soluble protein of the retinal interphotoreceptor matrix. Much is known concerning its role in the transport of retinoids between photoreceptor cells and pigment epithelium but little is known about its interactions with lipids. Here we have examined the physicochemical characteristics of a fatty acid binding site of IRBP using a set of fluorescent fatty acid analogs with an anthracene moiety attached at different positions along the hydrocarbon chain. The results show that fatty acids are bound in a hydrophobic environment as indicated by a blue shift in fluorescence maxima and by a increase in quantum yield of the bound ligand. There is a single specific fatty acid binding site for each molecule of IRBP with an apparent Kd = 3.6 x 10(-7) M. There is a nonradiative energy transfer from tryptophan residues to bound ligand. The interactions of IRBP and bound fatty acid are sensitive to denaturation by increasing concentrations of urea as judged by changes in nonradiative energy transfer efficiency and the quantum yield of bound probe. Quantum yields of bound fatty acid analogs varied with position of the fluorophore along the hydrocarbon chain and had the lowest values for the fluorophore located at the midpoint. Probing of the microenvironment of bound fluorophore with a quencher indicated a highly structured binding site.
光感受器间类视黄醇结合蛋白（IRBP）是一种140 kDa的糖脂蛋白，约占视网膜光感受器间基质可溶性蛋白的70%。关于它在光感受器细胞和色素上皮之间类视黄醇运输中的作用已了解很多，但对其与脂质的相互作用却知之甚少。在此，我们使用了一组荧光脂肪酸类似物，这些类似物在碳氢链的不同位置连接有蒽基部分，来研究IRBP脂肪酸结合位点的物理化学特性。结果表明，脂肪酸结合在疏水环境中，这可通过荧光最大值的蓝移和结合配体量子产率的增加来表明。每个IRBP分子有一个单一的特异性脂肪酸结合位点，表观解离常数Kd = 3.6 × 10⁻⁷ M。存在从色氨酸残基到结合配体的非辐射能量转移。根据非辐射能量转移效率和结合探针量子产率的变化判断，IRBP与结合脂肪酸的相互作用对尿素浓度增加导致的变性敏感。结合脂肪酸类似物的量子产率随荧光团在碳氢链上的位置而变化，位于中点的荧光团量子产率最低。用淬灭剂探测结合荧光团的微环境表明存在一个高度结构化的结合位点。
