Kanai M, Raz A, Goodman D S
J Clin Invest. 1968 Sep;47(9):2025-44. doi: 10.1172/JCI105889.
Vitamin A circulates in human plasma as retinol bound to a specific transport protein. This protein differs from the known low and high density plasma lipoproteins and has a hydrated density greater than 1.21. In order to study this protein, volunteers were injected intravenously with retinol-15-(14)C. Plasma was collected 1-3 days later, and the purification of retinol-binding protein (RBP) was monitored by assaying for (14)C and also by following the fluorescence of the protein-bound retinol. Purification of RBP was effected by the sequence: Cohn fractionation, chromatography on columns of Sephadex G-200 and diethylaminoethyl (DEAE)-Sephadex, preparative polyacrylamide gel electrophoresis, and finally chromatography on Sephadex G-100. These procedures resulted in a preparation of RBP which was at least 98% pure and which had been purified more than 1500-fold. Purified RBP has alpha(1) mobility on electrophoresis and has a molecular weight of approximately 21,000-22,000. There appears to be one binding site for retinol per molecule of RBP. Solutions of RBP are fluorescent (characteristic of retinol) and have ultraviolet absorption spectra with peaks at 330 mmu (resulting from the bound retinol) and at 280 mmu. There are no fatty acid or fatty acyl chains present in purified RBP. The usual concentration of RBP in plasma is of the order of 3-4 mg/100 ml. In plasma, RBP apparently circulates as a complex, together with another, larger protein with prealbumin mobility on electrophoresis. The RBP-prealbumin complex remains intact during Cohn fractionation and during chromatography on Sephadex and on DEAE-Sephadex columns. The complex dissociates during gel electrophoresis, permitting the isolation and subsequent purification of each of the components. The complex is again formed by mixing together solutions of the separated RBP and of prealbumin. Retinol transport in plasma thus appears to involve both a lipid-protein (retinol-RBP) interaction and a protein-protein (RBP-prealbumin) interaction.
维生素A在人体血浆中以与特定转运蛋白结合的视黄醇形式循环。这种蛋白质不同于已知的低密度和高密度血浆脂蛋白，其水合密度大于1.21。为了研究这种蛋白质，给志愿者静脉注射视黄醇-15-(14)C。1至3天后收集血浆，并通过检测(14)C以及跟踪蛋白质结合视黄醇的荧光来监测视黄醇结合蛋白（RBP）的纯化过程。RBP的纯化按以下顺序进行：科恩分级分离、在葡聚糖凝胶G - 200和二乙氨基乙基（DEAE）-葡聚糖柱上进行色谱分离、制备性聚丙烯酰胺凝胶电泳，最后在葡聚糖凝胶G - 100上进行色谱分离。这些步骤得到了一种RBP制剂，其纯度至少为98%，且已纯化了1500多倍。纯化的RBP在电泳中具有α(1)迁移率，分子量约为21,000 - 22,000。每个RBP分子似乎有一个视黄醇结合位点。RBP溶液具有荧光（视黄醇的特征），其紫外吸收光谱在330毫微米（由结合的视黄醇产生）和280毫微米处有峰值。纯化的RBP中不存在脂肪酸或脂肪酰链。血浆中RBP的通常浓度约为3 - 4毫克/100毫升。在血浆中，RBP显然以复合物的形式循环，与另一种在电泳中具有前白蛋白迁移率的更大蛋白质一起。RBP - 前白蛋白复合物在科恩分级分离以及在葡聚糖凝胶和DEAE - 葡聚糖柱上进行色谱分离的过程中保持完整。该复合物在凝胶电泳过程中解离，从而可以分离并随后纯化每种成分。通过将分离的RBP和前白蛋白的溶液混合在一起，又会再次形成复合物。因此，血浆中的视黄醇转运似乎涉及脂质 - 蛋白质（视黄醇 - RBP）相互作用和蛋白质 - 蛋白质（RBP - 前白蛋白）相互作用。
