Interaction between two dicarboxylate endogenous substances, bilirubin and an uremic toxin, 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid, on human serum albumin.

PURPOSE: Two dicarboxylate endogenous substances, bilirubin (BR) and 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF), have a very high affinity to human serum albumin (HSA). This study was undertaken to clarify the existence of a dicarboxylate binding site on HSA.
METHODS: Chemical modification, pH dependent binding and X-ray crystallographic analysis were performed to characterize these dicarboxylate binding sites.
RESULTS: It was found the binding behavior for dicarboxylates was different from typical site I ligands such as warfarin (WF) and phenylbutazone (PB) and that electrostatic interaction was an important factor for their binding to HSA. Moreover, His residues were considered to play an important role in pH dependent binding of dicarboxylic acids but in a different manner from the site I ligands. X-ray crystallography of CMPF and BR revealed the distances between the two carboxyl groups in their chemical structures were 5.854 A and 9.979 A, respectively. This difference may be reflected in pH dependent binding. Using fluorescent probe displacement, we attempted to identify the binding site for monocarboxylate derivatives of CMPF and investigated the role of individual carboxyl group in the recognition of the binding site. The results suggested two carboxyl groups were important for the specific binding of CMPF to site I.
CONCLUSIONS: The binding site for dicarboxylic acids is located in subdomain IIA, which includes site I, on the HSA molecule. Electrostatic interaction is an important driving force for binding to HSA.