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，与维生素结合 D及其代谢产物具有高亲和力，并反过来将它们输送到 靶器官和组织，导致观察到的嗜钙性 维生素D激素的性质。我们的目标是研究维他命 我们新近开发的光亲和法研究DBP的D-甾醇结合区 标记法。因此，我们计划对纯化的人进行光亲和标记 血清DBP(HDBP)与放射性标记的光亲和类似物~3H-25-ANE 25-羟基维生素D3，然后是蛋白质分解和化学切割，以及 一个或多个标记肽的分离。质谱分析 这些多肽片段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力 含有(I)不同部分光活性基团的25-OH-D3的类似物 母体分子，(Ii)具有不同长度的间隔臂，和(Iii) 带有卡宾前体的照片探针；并进行类似于 用~3H-25-烷描述的那些。这将导致明确的 HDBP中维生素D类固醇结合结构域的鉴定 结合部位内的接触点。

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.