BINDING OF HALOTHANE TO SERUM-ALBUMIN DEMONSTRATED USING TRYPTOPHAN FLUORESCENCE

Background:The site of action of general anesthesia remains controvers ial, but evidence in favor of specific protein target(s) is accumulati ng. Saturable binding of halothane to bovine serum albumin (BSA) has r ecently been reported using photoaffinity labeling and fluorine 19 nuc lear magnetic resonance spectroscopy, We report a new approach to stud y anesthetic binding to soluble proteins, based on native tryptophan f luorescence. Methods:Thymol-free halothane and fatty acid-free BSA wer e equilibrated in gas-tight Hamilton syringes and dispensed into stopp ered quartz cuvettes at predetermined dilutions. Steady-state fluoresc ence spectroscopy was used to study their interaction. Results:Halotha ne quenched the tryptophan fluorescence of BSA in a concentration-depe ndent, saturable manner with a dissociation constant = 1.8 +/- 0.2 mM and a Hill number = 1.0 +/- 0.1. The two optical isomers of halothane bound to BSA with equal affinity. The ability of halothane to quench B SA tryptophan fluorescence was markedly decreased at pH 3.0 (which cau ses full uncoiling of BSA), with loss of saturable binding, Diethyl et her displaced a portion of halothane from its binding sites, Circular dichroism spectroscopy revealed no significant effect of halothane or diethyl ether on the secondary structure of BSA. Conclusions: The resu lts suggest that halothane binds in hydrophobic domains containing try ptophan in BSA. This approach may prove useful for studying the intera ction of volatile anesthetics and proteins and has the advantage that the location of halothane in the protein is identified.