Binding of warfarin, salicylate, and diazepam to genetic variants of human serum albumin with known mutations.

Possible effects of single point mutations on the ligand-binding capabilities of human serum albumin (Alb) were investigated by studying the interactions between the strongly bound drugs warfarin, salicylate, and diazepam and five structurally characterized genetic variants of the protein. Equilibrium dialysis data, obtained with the variants and normal serum Alb, revealed pronounced reductions in high affinity binding of all three ligands to Alb Canterbury (313 Lys----Asn) and to Alb Parklands (365 Asp----His). By contrast, unchanged binding of the drugs was found in the case of Alb Verona (570 Glu----Lys). Different effects on binding were observed for the other two variants. Salicylate was the only drug bound with a lower affinity to Alb Niigata (269 Asp----Gly), whereas binding of both salicylate and diazepam to Alb Roma (321 Glu----Lys) were moderately reduced. In about half of the cases of diminished binding, the primary association constant was reduced by 1 order of magnitude, giving rise to an increase in the unbound fraction of the drugs of 500% or more at therapeutically relevant molar ratios of drug and protein. Changes in protein charge seem to be of only minor importance for reduced binding. More likely, conformational changes in the 313-365 region of the proteins are the main cause for diminished binding of these diverse ligands, which probably have different high affinity binding sites. The specific reduction in salicylate binding after modification of residue 269 may be due to conformational changes at or close to the salicylate binding site.