Canoa Pilar, Rivadulla Marcos L, Popplewell Jonathan, van Oosten René, Gómez Generosa, Fall Yagamare
Centro de Investigaciones Biomédicas (CINBIO), University of Vigo, Campus Lagoas Marcosende, 36310, Vigo, Spain.
Departamento de Química Orgánica, Facultad de Química and Instituto de Investigación Biomédica (IBI), University of Vigo, Campus Lagoas Marcosende, 36310, Vigo, Spain.
Anal Bioanal Chem. 2017 Apr;409(10):2547-2558. doi: 10.1007/s00216-017-0200-6. Epub 2017 Jan 23.
Vitamin D and its metabolites are lipophilic molecules with low aqueous solubility and must be transported bound to plasma carrier proteins, primarily to vitamin D binding protein (DBP). The biological functions of vitamin D metabolites are intimately dependent on the protein, hence the importance of determining their affinity for DBP. In this study, we developed a novel procedure for measuring the kinetic and equilibrium constants of human-DBP with vitamin D and three metabolites: 1,25-dihydroxyvitamin D [1,25(OH)D], 25-hydroxyvitamin D (25OHD) and 24,25-dihydroxyvitamin D [24,25(OH)D] by surface plasmon resonance (SPR). At the same time, five different analogues, synthetized in our laboratory, were evaluated in order to compare the affinity values with the metabolites. Real-time SPR measurements showed that 25OHD and 24,25(OH)D had higher affinity (0.3 μM) than 1,25(OH)D (5 μM), with the higher affinity of 25OHD and 24,25(OH)D due to dissociation constants 1 order of magnitude slower. In the case of the analogues, the affinity values were lower, with 1-hydroxy-25-nitro-vitamin D (NO-446), structurally closer to 1,25(OH)D, showing the highest value with a K of 50 μM. (24R)-1,25-dihydroxyvitamin-24-buthyl-28-norvitamin D (Bu-471) and (24R)-1,25-dihydroxyvitamin-24-phenyl-28-norvitamin D (Ph-491), structurally similar, had affinities of 180 and 170 μM, respectively. (22R,23E)-1-hydroxy-22-ethenyl-25-methoxy-23-dehydrovitamin D (MeO-455) and 1-hydroxy-20(R)-[5(S)-(2,2-dimethyltetrahydropyran-5-yl)]-22,23-dinor vitamin D (Oxan-429) had affinities of 310 and 100 μM, respectively. The binding of the metabolites and analogues was reversible allowing the rapid capture of data for replicates. The kinetic and equilibrium data for both the metabolites and the analogues fitted to the Langmuir model describing a 1:1 interaction. Graphical Abstract Label-free, real time binding study between vitamin D binding protein immmobilized on the surface of a SPR sensor chip and the vitamin D, metabolites and analogues passed over it as analytes.
维生素D及其代谢产物是亲脂性分子，水溶性低，必须与血浆载体蛋白结合运输，主要是与维生素D结合蛋白（DBP）结合。维生素D代谢产物的生物学功能密切依赖于该蛋白，因此确定它们与DBP的亲和力很重要。在本研究中，我们开发了一种新方法，通过表面等离子体共振（SPR）测量人DBP与维生素D及其三种代谢产物：1,25 - 二羟基维生素D [1,25(OH)D]、25 - 羟基维生素D（25OHD）和24,25 - 二羟基维生素D [24,25(OH)D]的动力学和平衡常数。同时，对我们实验室合成的五种不同类似物进行了评估，以便将亲和力值与代谢产物进行比较。实时SPR测量表明，25OHD和24,25(OH)D的亲和力（0.3 μM）高于1,25(OH)D（5 μM），25OHD和24,25(OH)D具有更高的亲和力是因为解离常数慢1个数量级。对于类似物，亲和力值较低，结构上更接近1,25(OH)D的1 - 羟基 - 25 - 硝基 - 维生素D（NO - 446）显示出最高值，K为50 μM。结构相似的(24R) - 1,25 - 二羟基维生素 - 24 - 丁基 - 28 - 去甲维生素D（Bu - 471）和(24R) - 1,25 - 二羟基维生素 - 24 - 苯基 - 28 - 去甲维生素D（Ph - 491）的亲和力分别为180和170 μM。(22R,23E) - 1 - 羟基 - 22 - 乙烯基 - 25 - 甲氧基 - 23 - 脱氢维生素D（MeO - 455）和1 - 羟基 - 20(R) - [5(S) - (2,2 - 二甲基四氢吡喃 - 5 - 基)] - 22,23 - 二去甲维生素D（Oxan - 429）的亲和力分别为310和100 μM。代谢产物和类似物的结合是可逆的，允许快速获取重复数据。代谢产物和类似物的动力学和平衡数据均符合描述1:1相互作用的朗缪尔模型。图形摘要固定在SPR传感器芯片表面的维生素D结合蛋白与作为分析物通过其上的维生素D、代谢产物和类似物之间的无标记实时结合研究。
