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Inhalational anesthetic agents are known to alter protein function, but the nature of the interactions underlying these effects remains poorly understood. We have used differential scanning calorimetry to study the effects of the anesthetic agent halothane on the thermally induced unfolding transition of bovine serum albumin. We find that halothane (0.6-10 mM) stabilizes the folded state of this protein, increasing its transition midpoint temperature from 62 to 71 degrees C. Binding of halothane to the native state of serum albumin thus outweighs any non-specific interactions between the thermally unfolded state of serum albumin and halothane in this concentration range. Based on the average enthalpy change DeltaH for unfolding of 170 kcal/mol, the increase from 62 to 71 degrees C corresponds to an additional Gibbs energy of stabilization (DeltaDeltaG) due to halothane of more than 4 kcal/mol. Analysis of the dependence of DeltaDeltaG on halothane concentration shows that thermal unfolding of a bovine serum albumin molecule is linked to the dissociation of about one halothane molecule at lower halothane concentrations and about six at higher halothane concentrations. Serum albumin is the first protein that has been shown to be stabilized by an inhalational anesthetic.

To understand further the weak molecular interactions between inhaled anesthetics and proteins, we studied the character and dynamic consequences of halothane binding to bovine serum albumin (BSA) and myoglobin using photoaffinity labeling and hydrogen-tritium exchange (HX). We find that halothane binds saturably and with submillimolar affinity to BSA, but either nonspecifically or with considerably lower affinity to myoglobin. Titration of halothane binding with guanidine hydrochloride suggested more protection of binding sites from solvent in BSA as compared with myoglobin. Protection factors for slowly exchanging albumin hydrogens are increased in a concentration-dependent manner by up to 27-fold with 10 mM halothane, whereas more rapidly exchanging groups of albumin hydrogens have either unaltered or decreased protection factors. Protection factors for slowly exchanging hydrogens in myoglobin are decreased by halothane, suggesting destabilization through binding to an intermediate or completely unfolded conformer. These results demonstrate the conformation dependence of halothane binding and clear dynamic consequences that correlate with the character of binding in these model proteins. Preferential binding and stabilization of different conformational states may underlie anesthetic-induced protein dysfunction, as well as provide an explanation for heterogeneity of action.