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Volume 272, Number 29, Issue of July 18, 1997 pp. 17961-17965
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Binding of the Volatile Anesthetic Chloroform to Albumin Demonstrated Using Tryptophan Fluorescence Quenching

(Received for publication, February 12, 1997, and in revised form, April 2, 1997)

From the Department of Anesthesia and the Johnson Research Foundation, University of Pennsylvania, Philadelphia, Pennsylvania 19104

The site(s) of action of the volatile general anesthetics remain(s) controversial, but evidence in favor of specific protein targets is accumulating. The techniques to measure directly volatile anesthetic binding to proteins are still under development. Further experience with the intrinsic protein fluorescence quenching approach to monitor anesthetic-protein complexation is reported using chloroform. Chloroform quenches the steady-state tryptophan fluorescence of bovine serum albumin (BSA) in a concentration-dependent, saturable manner with a K_d = 2.7 ± 0.2 mM. Tryptophan fluorescence lifetime analysis reveals that the majority of the quenching is due to a static mechanism, indicative of anesthetic binding. The ability of chloroform to quench BSA tryptophan fluorescence was decreased markedly in the presence of 50% 2,2,2-trifluoroethanol, which causes loss of tertiary structural contacts in BSA, indicating that protein conformation is crucial for anesthetic binding. Circular dichroism spectroscopy revealed no measurable effect of chloroform on the secondary structure of BSA. The results suggest that chloroform binds to subdomains IB and IIA in BSA, each of which contains a single tryptophan. Earlier work has shown that these sites are also occupied by halothane. The present study therefore provides experimental support for the theory that structurally distinct general anesthetics may occupy the same domains on protein targets.

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