Abstract
Electrostatic interactions have been proposed as a potentially important force for anesthetics and protein binding but have not yet been tested directly. In the present study, we used wild-type human serum albumin (HSA) and specific site-directed mutants as a native protein model to investigate the role of electrostatic interactions in halothane binding. Structural geometry analysis of the HSA-halothane complex predicted an absence of significant electrostatic interactions, and direct binding (tryptophan fluorescence and zonal elution chromatography) and stability experiments (hydrogen exchange) confirmed that loss of charge in the binding sites, by charged to uncharged mutations and by changing ionic strength of the buffer, generally increased both regional (tryptophan region) and global halothane/HSA affinity. The results indicate that electrostatic interactions (full charges) either do not contribute or diminish halothane binding to HSA, leaving only the more general hydrophobic and van der Waals forces as the major contributors to the binding interaction.
Highlights
Inhalational anesthetics can alter the activity of a wide variety of proteins, but the molecular nature of the interactions underlying the functional effect is still poorly understood
We used wild-type human serum albumin (HSA) and specific site-directed mutants as a native protein model to investigate the role of electrostatic interactions in halothane binding
Structural geometry analysis of the HSA-halothane complex predicted an absence of significant electrostatic interactions, and direct binding and stability experiments confirmed that loss of charge in the binding sites, by charged to uncharged mutations and by changing ionic strength of the buffer, generally increased both regional and global halothane/HSA affinity
Summary
Halothane (1-bromo-1-chloro-2,2,2-trifluoroethane) was obtained from Halocarbon Laboratories (Hackensack, NJ). Protein Data Bank file 1E7C by Swiss. K195 K199 F211 R218 R222 H242 R257 HLT 2001 HLT 2002 HLT 2004 HLT 2005 HLT 2006 HLT 2007. 9.1–12.1 4.5–8.4 3.5–7.4 5.3–8.4 9.8–14.3 4.3–9.8 11.4–14.7 11.6–13.8 12.5–14.6 11.1–13.9 8.9–11.4 5.3–8.4 23.5–26.5 ative and water in halothane were removed before use with an aluminum oxide column. [3H]OH was obtained from ICN (Costa Mesa, CA) at 100 mCi/ml. All other chemicals were reagent grade or better and were obtained from Sigma
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