Trp-145 is essential for the binding of 25-hydroxyvitamin D3 to human serum vitamin D-binding protein.
Chemical modification of specific amino acid residues in a protein has been a valuable tool in identifying amino acid residues that are responsible for ligand binding of a protein. In the present investigation, we targeted Trp and His residues in human serum vitamin D-binding protein (hDBP) by modifying them with specific chemical modifiers. We also evaluated the results of these modifications in the binding of 25-hydroxy[26(27)-3H]vitamin D3 ([3H]25-OH-D3) to hDBP. We observed a dose-dependent loss of binding activity by N-bromosuccinimide (specific for Trp). Similar results were observed with diethylpyrocarbonate (specific for His). Furthermore, loss of [3H]25-OH-D3-binding was protected by preincubation of hDBP samples with an excess of 25-hydroxyvitamin D3. These results strongly emphasized the importance of Trp (single residue at position 145) and 1 His residue (out of a total of 6) in the vitamin D sterol-binding by vitamin D-binding protein.
Department of Bioorganic and Protein Chemistry, Boston University School of Medicine, Massachusetts 02118.
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N
Swamy
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Brisson
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Ray
D001974:Bromosuccinimide; D002112:Calcifediol / Q000378:metabolism*; D006801:Humans; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000378:metabolism*; D014809:Vitamin D-Binding Protein / Q000097:blood / Q000378:metabolism*
D014809:Vitamin D-Binding Protein; D014364:Tryptophan; D001974:Bromosuccinimide; D002112:Calcifediol