title: Truncated human serum albumin retains general anaesthetic binding activity.
abstract: Multiple binding sites for anaesthetics in HSA (human serum albumin) make solution studies difficult to interpret. In the present study, we expressed the wild-type HSA domain 3 (wtHSAd3), a peptide with two known anaesthetic binding sites in a yeast expression system. We also expressed a site-directed mutant of domain 3 (Y411Wd3). The stability and secondary structure of the constructed fragments were determined by HX (hydrogen-tritium exchange) and CD spectroscopy. The binding of two general anaesthetics, 2-bromo-2-chloro-1,1,1-trifluoroethane and propofol, to wtHSAd3 and Y411Wd3 was determined using isothermal titration calorimetry, HX and intrinsic tryptophan fluorescence quenching. Although the expressed fragments are less stable than intact wtHSA as indicated by both CD and HX, they retain the secondary structure and anaesthetic-binding characteristics of an intact HSA molecule, but with fewer binding sites. Y411Wd3 had decreased affinity for propofol but not for 2-bromo-2-chloro-1,1,1-trifluoroethane, consistent with steric hindrance. Retention of structural features and anaesthetic binding properties with fewer binding sites in this truncated protein provide feasibility for using scaled-down models of otherwise intractable systems to gain an understanding of anaesthetic binding requirements and binding-stability relationships.
authors.affiliation: Department of Anesthesia, University of Pennsylvania Medical Center, 3400 Spruce Street, 7 Dulles, Philadelphia, PA 19104-4283, USA.
authors.forename: Renyu
authors.initials: R
authors.lastname: Liu
authors.forename: Jinsheng
authors.initials: J
authors.lastname: Yang
authors.forename: Chung-Eun
authors.initials: CE
authors.lastname: Ha
authors.forename: Nadhipuram V
authors.initials: NV
authors.lastname: Bhagavan
authors.forename: Roderic G
authors.initials: RG
authors.lastname: Eckenhoff
mesh_terms: D018681:Anesthetics, General / Q000737:chemistry*; D001665:Binding Sites; D006221:Halothane / Q000737:chemistry*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D015742:Propofol / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012709:Serum Albumin / Q000737:chemistry*; D013816:Thermodynamics
chemical_list: D018681:Anesthetics, General; D012709:Serum Albumin; D006221:Halothane; D015742:Propofol