PROBING THE BINDING SITE IN VITAMIN D BINDING PROTEIN

维生素D结合蛋白（DBP）是一种主要的血清蛋白， D及其代谢产物具有高亲和力，并反过来将它们转运到 靶器官和组织，导致观察到的钙离子依赖性 维生素D激素的性质。 我们的目标是探索维生素 D甾醇结合结构域的DBP 标记法 因此，我们计划光亲和标记纯化的人 血清DBP（hDBP）与3 H-25-ANE（放射性标记的光亲和类似物） 25-羟基维生素D3，然后进行蛋白水解和化学裂解，和 分离标记的肽。 质谱分析 这种肽片段的分析将使我们能够定义维生素 hDBP中的D-结合位点。 我们还将开发额外的光亲和性 25-OH-D3的类似物，其含有（i）在25-OH-D3的不同部分的光活性基团， 母体分子，（ii）具有不同长度的间隔臂，和（iii） 光探针与卡宾前体;并进行类似的研究， 用3 H-25-ANE描述的那些。 这将导致明确的 hDBP中维生素D甾醇结合结构域的鉴定以及 结合位点内的接触点。

Vitamin D binding protein (DBP), a major serum protein, binds to vitamin D and its metabolites with high affinity, and in turn, transports them to target organs and tissues, leading to the observed calciotropic properties of vitamin D hormone. Our objectives are to probe the vitamin D sterol-binding domain of DBP by our recently developed photoaffinity labelling method. Thus, we plan to photoaffinity label purified human serum DBP (hDBP) with 3H-25-ANE, a radiolabelled photoaffinity analog of 25-hydroxyvitamin D3, followed by proteolytic and chemical cleavages, and isolation of the labelled peptide/peptides. Mass spectrometric analysis of this peptide fragment/fragments will enable us to define the vitamin D-binding site in hDBP. We will also develop additional photoaffinity analogs of 25-OH-D3 containing (i) photoactive groups at various part of the parent molecule, (ii) spacer arm having different lengths, and (iii) photo probes with carbene precursors; and carry out studies similar to those described with 3H-25-ANE. This will lead to unequivocal identification of the vitamin D sterol-binding domain in hDBP as well as points of contact within the binding site.