Stoichiometry and Binding Site Analysis of Ligand-Human Serum Albumin Complex by Photoaffinity Labeling and Mass Spectrometry
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概要
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Using mass spectrometry and photoaffinity labeling, we have analyzed the binding of ligands to human serum albumin (HSA). Human serum albumin was selected as the protein, the site photolabeled with the photoaffinity labeling reagent FNAK {(+)-(R)-3,4-dihydro-2-[5-methoxy-2-[3-[N-methyl-N-[2-(3-azidophenoxy)-ethyl]amino]propoxyl]phenyl]-4-methyl-2H-1,4-benzothiazin-3-(4H)-one} was analyzed, and Lys residues at two positions (major: Lys-414 and minor: Lys-541) were found to be the sites photolabeled with FNAK. Based on the results of photolabeling, we proposed a model of the FNAK-HSA complex [K. Kawahara et al., Biochem. J., 363, 223 (2002)]. In this study, for the purpose of clarifying the binding site of the photoaffinity labeling reagent FNAK on HSA molecule, stoichiometry of FNAK in HSA was analyzed in detail by nanoelectrospray ionization mass spectrometry (nanoESI MS), and stoichiometry of FNAK and HSA was found to be 1 : 1. On the basis of these results, a model of HSA-FNAK complex was constructed by the molecular dynamics calculation method. The model strongly suggested that the Myr-3 and Myr-4 binding pockets in subdomain IIIA was the FNAK binding site.
- 日本質量分析学会の論文
- 2005-02-01
著者
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MASUDA KATSUYOSHI
Suntory Institute for Bioorganic Research
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Nakayama Hitoshi
Department of Pharmacology, Nara Medical University School of Medicine, Japan
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ISHIGURO Masaji
Suntory Institute for Bioorganic Research
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Nakayama Hitoshi
Department Of Agricultural Chemistry College Of Agriculture University Of Osaka Prefecture
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Kuniyasu Akihiko
Department Of Biofunctional Chemistry Faculty Of Pharmaceutical Sciences Kumamoto University
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Kuniyasu Akihiko
Department Of Molecular Cell Function Graduate School Of Medical And Pharmaceutical Sciences Kumamot
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KAWAHARA Kohichi
Department of Molecular Cell Function, Graduate School of Medical and Pharmaceutical Sciences, Kumam
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Nakayama Hitoshi
Department Of Molecular Cell Function Graduate School Of Medical And Pharmaceutical Sciences Kumamot
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