title: Determining binding sites of drugs on human serum albumin using FIA-QCM.
abstract: A simple and effective method was developed for determining binding sites of drugs on human serum albumin (HSA) by independent binding or competitive displacement of bilirubin using flow injection analysis-quartz crystal microbalance (FIA-QCM) system. Both independent and competitive bindings were entirely monitored in real time. Bilirubin as a site I-binding ligand was pre-bound to HSA sensor so as to occupy the drug-binding site I. When the model site II-binding drugs (ibuprofen, ketoprofen and flurbiprofen) were injected into the bilirubin pre-bound HSA system, the frequency continuously decreased by 6 Hz, 4 Hz and 5 Hz, respectively, which was the same as that of their individual binding to HSA sensor. It indicated that the drug binding to site II was independent and did not interfere with bilirubin binding. However, when the model site I-binding drugs (iodipamide and magnesium salicylate) were introduced into the system, the frequency remained unchanged in the initial several minutes and then rapidly decreased by 4 Hz for iodipamide and increased by 4 Hz for magnesium salicylate. This phenomenon revealed site I-binding drugs competitively bound to HSA against bilirubin and displaced the pre-bound bilirubin. The results demonstrate FIA-QCM can be a valid approach for monitoring the dynamic interaction between drugs and HSA in real time further identifying drug-binding sites without the need of labels.
authors.affiliation: Beijing National Laboratory for Molecular Sciences, Laboratory of Analytical Chemistry for Life Science, Institute of Chemistry, Chinese Academy of Sciences, Beijing, China.
authors.forename: Qundan
authors.initials: Q
authors.lastname: Zhang
authors.forename: Yanyan
authors.initials: Y
authors.lastname: Huang
authors.forename: Rui
authors.initials: R
authors.lastname: Zhao
authors.forename: Guoquan
authors.initials: G
authors.lastname: Liu
authors.forename: Yi
authors.initials: Y
authors.lastname: Chen
mesh_terms: D001663:Bilirubin / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D001667:Binding, Competitive; D015374:Biosensing Techniques / Q000379:methods*; D004566:Electrodes; D017022:Flow Injection Analysis / Q000379:methods*; D006801:Humans; D004364:Pharmaceutical Preparations / Q000378:metabolism*; D011485:Protein Binding; D011791:Quartz / Q000737:chemistry; D012709:Serum Albumin / Q000737:chemistry* / Q000378:metabolism
chemical_list: D004364:Pharmaceutical Preparations; D012709:Serum Albumin; D011791:Quartz; D001663:Bilirubin