Location of drug binding sites on human serum albumin.

Abstract

The location of Site I on the human serum albumin (HSA) molecule was investigated.Naphthol yellow- S (NY-S) was used as a representative ligand for Site I, since the bilirubin-displacing effect of NY-S was similar to that of Site I drugs such as warfarin and phenylbutazone.NY-S was bound to HSA with at least three classes of binding sites. The binding parameters of the primary class were one binding site (n1=1) and a binding constant (K1) of >107M-1, and the complex at equimolar ratio of dye to albumin exhibited metachromasy. The intrinsic fluorescence intensity, attributed to the single tryptophan residue (position 214), decreased significantly in this complex. Fragments A and C (residues 299-585 and 124-298, respectively, in the amino acid sequence of HSA) obtained by cyanogen bromide cleavage of HSA showed NY-S-binding ability.The binding of NY-S to fragment A or C aused the same metachromasy as seen with NY-S-HSA complex. Fragment C is markedly hydrophobic and contains the single tryptophan residue. The binding of NY-S to HSA was diminished by the chemical modification of lysine residues with pyridoxal 5'-phosphate (PLP). Further, lysine residues modified by PLP existed in fragment C.These results suggest that the primary binding site of NY-S, which corresponds to Site I, is located in the fragment C region of the HSA molecule.