tailieunhanh - Báo cáo khoa hoc : Biological characteristics of two lysines on human serum albumin in the high-affinity binding of 4Z,15Z-bilirubin-IXa revealed by phage display

4Z,15Z-bilirubin-IXa(4Z,15Z-BR), an endogenous compound that is spar-ingly soluble in water, binds human serum albumin (HSA) with high affin-ity in a flexible manner. A phage library displaying recombinant HSA domain II was constructed, after three rounds of panning against immobi-lized 4Z,15Z-BR | IFEBS Journal Biological characteristics of two lysines on human serum albumin in the high-affinity binding of 4Z 15Z-bilirubin-IXa revealed by phage display Ai Minomo1 Yu Ishima1 Ulrich Kragh-Hansen2 Victor T. G. Chuang3 Makiyo Uchida4 Kazuaki Taguchi1 Hiroshi Watanabe1 Toru Maruyama1 Hiroshi Morioka4 and Masaki Otagiri1 5 1 Department of Biopharmaceutics Graduate Schoolof PharmaceuticalSciences Kumamoto University Japan 2 Department of MedicalBiochemistry University of Aarhus Denmark 3 Schoolof Pharmacy Curtin Health Innovation Research Institute Curtin University Perth Australia 4 Department of Analyticaland BiophysicalChemistry Graduate Schoolof PharmaceuticalSciences Kumamoto University Japan 5 Drug Delivery System Research Institute Faculty of PharmaceuticalSciences Sojo University Kumamoto Japan Keywords bilirubin domain II high-affinity binding human serum albumin phage display Correspondence M. Otagiri Faculty of Pharmaceutical Sciences Sojo University 1-22-4 Ikeda Kumamoto-shi Kumamoto 860-0082 Japan Fax 81 96 362 7690 Tel 81 96 371 4150 E-mail otagirim@ Received 12 April 2011 revised 30 June 2011 accepted 24 August 2011 doi 4Z 15Z-bilirubin-IXa 4Z 15Z-BR an endogenous compound that is sparingly soluble in water binds human serum albumin HSA with high affinity in a flexible manner. A phage library displaying recombinant HSA domain II was constructed after three rounds of panning against immobilized 4Z 15Z-BR and eight clones with high affinity for the pigment were found to contain conserved basic residues such as lysine or arginine at positions 195 and 199. The wild type and two mutants K195A and K199A of whole HSA as well as stand-alone domain II were expressed in Pichia pastoris for ligand-binding studies. The binding of 4Z 15Z-BR to the K195A and K199A mutants was decreased in both whole HSA and the domain II proteins. The P-helicity conformer P-form of 4Z 15Z-BR was found to preferentially bind to the .