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.