The role of electrostatic interactions in human serum albumin binding and stabilization by halothane

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.