Binding of halothane to serum albumin demonstrated using tryptophan fluorescence.
BACKGROUND
The site of action of general anesthesia remains controversial, but evidence in favor of specific protein target(s) is accumulating. Saturable binding of halothane to bovine serum albumin (BSA) has recently been reported using photoaffinity labeling and fluorine 19 nuclear magnetic resonance spectroscopy. We report a new approach to study anesthetic binding to soluble proteins, based on native tryptophan fluorescence.


METHODS
Thymol-free halothane and fatty acid-free BSA were equilibrated in gas-tight Hamilton syringes and dispensed into stoppered quartz cuvettes at predetermined dilutions. Steady-state fluorescence spectroscopy was used to study their interaction.


RESULTS
Halothane quenched the tryptophan fluorescence of BSA in a concentration-dependent, 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 BSA tryptophan fluorescence was markedly decreased at pH 3.0 (which causes full uncoiling of BSA), with loss of saturable binding. Diethyl ether 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 results suggest that halothane binds in hydrophobic domains containing tryptophan in BSA. This approach may prove useful for studying the interaction of volatile anesthetics and proteins and has the advantage that the location of halothane in the protein is identified.
Johansson
J S
JS
Department of Anesthesiology, University of Pennsylvania, Philadelphia 19104-4283, USA.
Eckenhoff
R G
RG
Dutton
P L
PL
D005453:Fluorescence; D006221:Halothane / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D011485:Protein Binding; D012709:Serum Albumin / Q000378:metabolism*; D014364:Tryptophan
D012709:Serum Albumin; D014364:Tryptophan; D006221:Halothane