tailieunhanh - Báo cáo khoa học: Binding of the volatile general anesthetics halothane and isoflurane to a mammalian b-barrel protein

A molecular understanding of volatile anesthetic mechanisms of action will require structural descriptions of anesthetic–protein complexes. Porcine odorant binding protein is a 157 residue member of the lipocalin family that features a large b-barrel internal cavity (515 ± 30 A ˚ 3 ) lined predomin-antly by aromatic and aliphatic residues. Halothane binding to theb-barrel cavity was determined using fluorescence quenching of Trp16, and a com-petitive binding assay with 1-aminoanthracene. In addition, the binding of halothane and isoflurane were characterized thermodynamically using iso-thermal titration calorimetry | ềFEBS Journal Binding of the volatile general anesthetics halothane and isoflurane to a mammalian 0-barrel protein Jonas S. Johansson1 2 4 Gavin A. Manderson1 Roberto Ramoni5 Stefano Grolli5 and Roderic G. Eckenhoff1 3 1 Department of Anesthesia University of Pennsylvania Philadelphia PA USA 2 Department of Biochemistry and Biophysics University of Pennsylvania Philadelphia PA USA 3 Department of Physiology University of Pennsylvania Philadelphia PA USA 4 Johnson Research Foundation University of Pennsylvania Philadelphia PA USA 5 Dipartimento di Produzioni Animali Biotecnologie Veterinarie Qualita e Sicurezza degli Alimenti University di Parma Parma Italy Keywords anesthetic-protein interaction halothane isoflurane isothermal titration calorimetry porcine odorant binding protein Correspondence J. S. Johansson 319C John Morgan Building University of Pennsylvania 3620 Hamilton Walk Philadelphia PA 19104 USA Fax 1 215 349 5078 Tel 1 215 349 5472 E-mail JohanssJ@ Received 18 October 2004 revised 19 November 2004 accepted 24 November 2004 doi A molecular understanding of volatile anesthetic mechanisms of action will require structural descriptions of anesthetic-protein complexes. Porcine odorant binding protein is a 157 residue member of the lipocalin family that features a large b-barrel internal cavity 515 30 A3 lined predominantly by aromatic and aliphatic residues. Halothane binding to the b-barrel cavity was determined using fluorescence quenching of Trp16 and a competitive binding assay with 1-aminoanthracene. In addition the binding of halothane and isoflurane were characterized thermodynamically using isothermal titration calorimetry. Hydrogen exchange was used to evaluate the effects of bound halothane and isoflurane on global protein dynamics. Halothane bound to the cavity in the b-barrel of porcine odorant binding protein with dissociation constants of and mM determined using fluorescence .