The enantioselective immunoaffinity extraction of an optically active ibuprofen-modified peptide fragment

Acyl glucuronides are known to produce the covalently bound protein adducts which may be the cause of hypersensitivity and toxic responses to acidic d rugs. The structural analysis of the drug-protein adducts is therefore need ed. From this point of view, we developed an enantioselective immunoaffinit y extraction method, which employs an immobilized antibody to specifically isolate peptide fragments that have been modified with optically active ibu profen. Rabbits were immunized with (S)-ibuprofen coupled to bovine serum a lbumin through a beta -alanine group. The elicited antibody strongly recogn izes the asymmetric center and the isobutylphenyl moiety of (S)-ibuprofen a nd its conjugates but has a low affinity for their anti podes. A 0.5-mL ali quot of the immunosorbent (11.5 mg of IgG/mL gel) prepared by immobilizatio n of the antibody was capable of retaining up to 1 mug of (S)-ibuprofen. Wh en a mixture of substance P with (R)- and (S)-ibuprofen-modified substance P was loaded on the immunosorbent, the (S)-ibuprofen-modified substance P w as selectively retained. The modified peptide was quantitatively recovered by elution with 10 mM ammonium acetate buffer (pH 5.0)/methanol (5:95, v/v) . The proposed method would be useful for the structural characterization o f optically active ibuprofen-modified human serum albumin. (C) 2001 Academi c Press.