Since acyl glucuronides are known to undergo deconjugation, especially in the presence of human serum albumin (HSA), only a few reports have described their reversible binding to plasma proteins. The aim of this study was to investigate the reversible binding of R and S ketoprofen glucuronides to HSA by a rapid technique, such as ultraviolet circular dichroism. Binding of R ketoprofen glucuronide only induced an extrinsic Cotton effect at 340 nm. Scatchard plot analysis revealed that R ketoprofen and its glucuronide are bound to one site of albumin with an association constant of 28.1 x 10(4) and 6.1 x 10(4) M-1, respectively. Modification of one tyrosine residue by diisopropylfluorophosphate prevented the access of ligands to sites I and II of albumin, and also fully inhibited the binding of R ketoprofen and that of its conjugate. Displacement experiments with specific probes of albumin binding sites suggested that R ketoprofen and the glucuronide are bound to site II rather than site I. However, R ketoprofen was not displaced by its conjugate. S ketoprofen glucuronide is also bound to HSA, since it decreased the binding of the antipode conjugate. However, the binding of this metabolite to albumin did not induce an extrinsic Cotton effect large enough to determine the binding constants. D-Glucuronic acid did not bind to sites I or II of albumin. This moiety is likely responsible for the lower affinity of HSA for the R ketoprofen glucuronide when compared to that for R ketoprofen, due to the hydrophilicity and/or the bulkiness of this group.