Low-affinity analytical chromatography for measuring inhaled anesthetic binding to isolated proteins.
The direct measure of volatile anesthetic binding to protein is complicated by weak affinity and therefore rapid kinetics. Consequently, several puted targets for these clinically important drugs have only functional data to support a direct mode of action. While several methods for measuring some aspects of binding are available, all have significant limitations. We introduce the use of analytical chromatography for the purpose of directly measuring volatile anesthetic binding to protein, and show that it can provide estimates of both affinity and stoichiometry for proteins that can be obtained in fairly high purity and mass. Using this approach we characterize halothane binding to serum albumin as low affinity and multisite, and to myoglobin or cytochrome C as strictly nonspecific. This approach will be useful in directly characterizing equilibrium, solution binding to isolated proteins in preparation for more time-consuming methods with structural resolution.
Chan
Kin
K
Department of Anesthesia, University of Pennsylvania Health System, Philadelphia, Pennsylvania 19104, USA.
Meng
Qing C
QC
Johansson
Jonas S
JS
Eckenhoff
Roderic G
RG
D018685:Anesthetics, Inhalation / Q000032:analysis / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002846:Chromatography, Affinity / Q000379:methods*; D003574:Cytochrome c Group / Q000302:isolation & purification / Q000378:metabolism*; D006221:Halothane / Q000032:analysis* / Q000378:metabolism; D006736:Horses; D009211:Myoglobin / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D012710:Serum Albumin, Bovine / Q000302:isolation & purification / Q000378:metabolism*
D018685:Anesthetics, Inhalation; D003574:Cytochrome c Group; D009211:Myoglobin; D012710:Serum Albumin, Bovine; D006221:Halothane