High-affinity binding of warfarin, salicylate and diazepam to natural mutants of human serum albumin modified in the C-terminal end.

High-affinity binding of warfarin, salicylate and diazepam to four natural mutants of human serum albumin was studied by equilibrium dialysis at pH 7.4. The mutants Alb Milano Fast and Alb Vanves possess single amino acid substitutions close to the C-terminus, namely 573 Lys-->Glu and 574 Lys-->Asn, respectively. By contrast, Alb Catania and Alb Venezia are chain termination mutants in which several amino acids have been changed or deleted. Binding of warfarin to the variants was lower than binding to normal (wild-type) albumin (Alb A). The association constants were 73% (Alb Milano Fast, Alb Vanves and Alb Catania) or 67% (Alb Venezia) of that determined for Alb A. The results obtained with salicylate were more dependent on the type of mutation. The constants were either comparable to the normal value (Alb Catania) or reduced to 64% (Alb Milano Fast), 71% (Alb Vanves) or 43% (Alb Venezia) of that value. Diazepam binding to the variants was normal, except for binding to Alb Venezia in which case the association constant was reduced to 76% of that calculated for Alb A. The results are in accordance with the view that warfarin, salicylate and diazepam bind to three different high-affinity sites. It is proposed that the sites for warfarin and salicylate are situated rather close to each other in domain II, and that these high-affinity sites are relatively susceptible to conformational changes of the protein. By contrast, the primary diazepam site is placed closer to, or within, domain III of albumin and seems to be less affected by conformational changes in the protein molecule.