Free
Correspondence: reply  |   December 1995
Binding of Halothane to Serum Albumin: Relevance to Theories of Narcosis
Article Information
Pharmacology
Correspondence: reply   |   December 1995
Binding of Halothane to Serum Albumin: Relevance to Theories of Narcosis
Anesthesiology 12 1995, Vol.83, No Pagination Specified. doi:
Anesthesiology 12 1995, Vol.83, No Pagination Specified. doi:
In Reply:--Eger and Koblin raise several issues concerning the validity of albumin as a model to study mechanisms of inhalational anesthetic action and the relevance of the concentrations both of halothane and diethyl ether used in our studies. The principal aim of our communication 1was to describe an alternative and useful approach to determining anesthetic-protein interactions. Albumin was selected to compare the tryptophan fluorescence quenching method with the previous studies on anesthetic binding to this protein, which used19F-NMR 2and photoaffinity labeling. 3Our experiments were performed at 25 degrees Celsius to permit comparison with the earlier studies 2-3conducted at similar temperatures and because the direct information available 4suggests that, at least over the temperature range from 5 degrees Celsius to 20 degrees Celsius, volatile general anesthetic binding to protein targets only changes by 20–30%.
The measured dissociation constants of halothane and diethyl ether determined using quenching of tryptophan fluorescence 1are greater than those required to maintain the anesthetic state in animals. It would be remarkable if the anesthetic binding sites on albumin exactly reproduced the clinical pharmacology of the inhaled general anesthetics. However, the fact that these tryptophan domains bind anesthetics within an order of magnitude of their clinical ED50's suggests that they may share some characteristics with the physiologically relevant sites. Although this was not the primary goal of the work, this is a satisfactory first approximation, because a tenfold change in affinity only corresponds to a binding energy of 1.4 kcal/mol (comparable to a single hydrogen bond 5). Our study opens the door methodologically for a molecular description of anesthetic binding sites in proteins: For example, one avenue would be the use of site-directed mutagenesis of the albumin sites to conduct a structure-affinity study with a variety of volatile anesthetic molecules.
In further response to the points made by Eger and Koblin concerning the weakness of the binding and the failure to reproduce the relative order of potencies seen clinically, it should be emphasized that it remains to be established that these structurally diverse molecules act at a single locus. Furthermore, it is far from clear that clinical EC50values must correspond directly to experimental dissociation constants. These questions remain unanswered, because the methodology to directly study volatile anesthetic binding has been unavailable until recently and is under development. Binding studies accounting for anesthetic desolvation and the formation of interactions with protein sites with a focus on molecular structure are needed. Using our approach 1and other techniques, 2-3the answers to these questions are forthcoming.
Jonas S. Johansson, M.D., Ph.D., Roderic G. Eckenhoff, M.D., P. Leslie Dutton, F.R.S., Departments of Anesthesiology and the Johnson Research Foundation, Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania 19104.
REFERENCES
Johansson JS, Eckenhoff RG, Dutton PL: Binding of halothane to serum albumin demonstrated using tryptophan fluorescence. ANESTHESIOLOGY 83:316-324, 1995.
Dubois BW, Cherian SF, Evers AS: Volatile anesthetics compete for common binding sites on bovine serum albumin: A 19F-NMR study. Proc Natl Acad Sci USA 90:6478-6482, 1993.
Eckenhoff RG, Shuman H: Halothane binding to soluble proteins determined by photoaffinity labeling. ANESTHESIOLOGY 79:96-106, 1993.
Dickinson R, Franks NP, Lieb WR: Thermodynamics of anesthetic/protein interactions: Temperature studies on firefly luciferase. Biophys J 64:1264-1271, 1993.
Shirley BA, Stanssens P, Hahn U, Pace CN: Contribution of hydrogen bonding to the conformational stability of ribonuclease T1. Biochemistry 31:725-732, 1992.