Binding of a chiral drug to a protein: an investigation of the 2-(3-benzoylphenyl)propionic acid/bovine serum albumin system by circular dichroism and fluorescence

Abstract

A combined approach using global analysis of circular dichroism multiwavelength data and time resolved fluorescence was applied to investigate the interaction of R-(-)- and S-(+)-ketoprofen with bovine serum albumin in buffer solution at neutral pH. A characterization of the most stable drug : protein adducts of 1 : 1 and 2 : 1 stoichiometry, as individual chemical species, was obtained. The stability constants and the absolute circular dichroism spectra of the diastereomeric complexes were determined. The spectra of the 1 : 1 conjugates are opposite in sign, those of the 2 : 1 complexes are both negative, but different in shape from each other (peaks at 358 and 342 nm for S-(+)- and R-(-)-ketoprofen, respectively). A tryptophan residue was shown to be involved in the binding of the drug, in the primary site for the R-(-) and in the secondary site for the S-(+) enantiomer, thereby showing that chiral recognition by the protein causes the site of highest affinity being not the same for both optical antipodes.

Diastereoselectivity and Site Dependency in the Photochemistry of Ketoprofen in the Bovine Serum Albumin Matrix†

“…In the analysis of the sets of spectra relevant to the R(–)‐enantiomer at 1.5 × 10 ‐5 and 2.4 × 10 ‐4 M BSA concentration, convergence in the minimization procedure was obtained by imposing as constraints the absolute CD spectra of the 1:l and the 2:l complexes, determined by global analysis of the experiment at 4.5 × 10 ‐5 M BSA concentration (Fig. 2a, b) (14) *.…”

“…These data can be discussed in more detail in the light of the results of a study, previously performed, of the time‐resolved tryptophan fluorescence in the KP/BSA system (14), which indicated that the sites involved in the binding of KP enantiomers were Site I in Subdomain IIA and Site II in Subdomain IIIA (site names from Sudlow [20] and domain assignment from the X‐ray structure of the homologue HSA [21,22]), the 1:l complexation of R(–)‐KP occurring in Subdomain IIA and that of S(+)‐KP in Subdomain IIIA. Thus for each enantiomer the site of highest affinity turns out to be the most protective.…”

“…R(–)‐2‐(3‐benzoylphenyl)propionic acid was prepared according to a biocatalytic procedure consisting of three sequential steps and exploiting lipase from Candida antarctica , of known R stereopreference, adsorbed on acrylic resin (Novoyme 435, Sigma‐Aldrich). A detailed description of the preparation procedure has already been given (14).…”

“…We have recently examined the chiral recognition of KP enantiomers by BSA with the use of circular dichroism (CD) and fluorescence (14). This study, performed in the KP/BSA molar ratio range from 0.4 to 2.2, evidenced enantioselective binding and allowed us to achieve a characterization of the two main protein‐binding sites for each enantiomer.…”

Chiral recognition of metalaxyl enantiomers by human serum albumin: evidence from molecular modeling and photophysical approach

“…3 Interestingly, HSA is also known to possess the supreme enantioselectivity among human plasma proteins. Monti et al [11][12][13][14][15] studied the binding of (R)-/ (S)-ketoprofen to serum albumin in detail and stated that site I in subdomain IIA as the site of the highest affinity for (R)-ketoprofen and the secondary one for (S)-ketoprofen, whereas site II in subdomain IIIA denotes the main binding site for (S)-ketoprofen and the secondary site for (R)-ketoprofen. For this case, the sole enantiomers serve as disparate substances, their absorption, distribution, metabolism, excretion, and toxicity diverging in essence.…”