Binding of the volatile anesthetic chloroform to albumin demonstrated using tryptophan fluorescence quenching.
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 Kd = 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.
Johansson
J S
JS
Department of Anesthesia and the Johnson Research Foundation, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA. johansso@mail.med.upenn.edu
D018685:Anesthetics, Inhalation; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002725:Chloroform / Q000097:blood*; D002942:Circular Dichroism; D006801:Humans; D007700:Kinetics; D011485:Protein Binding; D011487:Protein Conformation; D012709:Serum Albumin / Q000737:chemistry / Q000378:metabolism*; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014364:Tryptophan
D018685:Anesthetics, Inhalation; D012709:Serum Albumin; D012710:Serum Albumin, Bovine; D002725:Chloroform; D014364:Tryptophan