Topology Analysis of Drug Binding Sites on Serum Proteins using Photoaffinity Labeling Techniques

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Arylpropionic acid NSAIDs and benzodiazepines are known to bind to site II of human serum albumin (HAS). However, information on the amino acids of the binding site that interact with these drugs is still insufficient. Ketoprofen (KP), an arylpropionic acid NSAID possessing benzophenone moiety and flunitrazepam (FNZP), a benzodiazepine with nitrophenyl moiety were used as photoaffinity labeling agents to define the microenvironment of site II on HAS. CNBr fragments of the [ィイD114ィエD1C]KP photolabeled HAS of about 14 kDa and 9 kDa incorporated radioactivity. Competition experiment showed that the 14 kDa band was the specifically photolabeled band. The 14 kDa peptide was redigested with Achromobacter lyticus protease I (AP I) and then separated with capillary HPLC. The amino acid sequence of the [ィイD114ィエD1C]KP photolabeled peptide was concluded to be XCTESLVNRR, which corresponded to 476C - 500K of HAS. This region of HAS was reported to be part of the site II hydrophobic binding pocket. Benzodiazepine (BDZ) drugs are known to bind to both human serum albumin (HAS) and alphal-acid glycoprotein (AGP). It is generally accepted that benzodiazepine drugs bind to subdomain III A (site II) of HAS. CNBr fragment of about 20 kDa of the [ィイD13ィエD1H]FNZP photolabeled HAS incorporated most of the radioactivity while the major photolabeled band of AGP seemed to be the N-terminus CNBr fragment The 20 kDa band radioactivity decreased sharply when HAS was photolabeled with FNZP in the presence of myristic acid but not in the presence of diazepam. The reverse displacement effect was observed for the N-terminus band of AGP. Further fluorescent probe displacement experiment showed that FNZP failed to displace the site II probe dansylsarcosine. The key position in the BDZ molecular structure that governs the binding affinity to site II of HAS was concluded to be position 7 of ring A.