Anesthetic stabilization of protein intermediates: myoglobin and halothane.
Halothane, an inhaled anesthetic, destabilizes the folded structure of myoglobin. To determine whether this is due to preferential interactions with less stable folded conformers of myoglobin versus the completely unfolded state, we used photoaffinity labeling, hydrogen exchange, fluorescence spectroscopy, and circular dichroism spectroscopy. Apomyoglobin was used as a model of a less stable conformer of myoglobin. Halothane destabilizes myoglobin and binds with low affinity and stoichiometry but stabilizes and binds with higher affinity to apomyoglobin. The same halothane concentration has no effect on cytochrome c stability. The apomyoglobin/halothane complex is favored at pH 6.5 as compared to pH 4.5 or pH 2.5. Halothane photoincorporates into several sites in apomyoglobin, some allosteric to the heme pocket. Guanidinium unfolding of myoglobin, monitored by CD spectroscopy, shows destabilization at less than 1.3 M Gdm but stabilization at greater than 1.3 M Gdm, consistent with the hypothesis that less stable conformers of myoglobin bind halothane preferentially. We suggest the structural feature underlying preferential binding to less stable conformers is an enlarged cavity volume distribution, since myoglobin has several intermediate-sized cavities, while cytochrome c is more well packed and has no cavities detected by GRASP. Specific binding to less stable intermediates may underlie anesthetic potentiation of protein activity.
Eckenhoff
R G
RG
Department of Anesthesia, University of Pennsylvania Medical School, Philadelphia, Pennsylvania 19104, USA. reckenho@mail.med.upenn.edu
Pidikiti
R
R
Reddy
K S
KS
D000595:Amino Acid Sequence; D018685:Anesthetics, Inhalation / Q000737:chemistry; D000818:Animals; D001059:Apoproteins / Q000737:chemistry*; D001665:Binding Sites; D002250:Carbon Radioisotopes; D003574:Cytochrome c Group / Q000737:chemistry; D019791:Guanidine; D006221:Halothane / Q000737:chemistry*; D006736:Horses; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009211:Myoglobin / Q000737:chemistry*; D010446:Peptide Fragments / Q000737:chemistry; D019920:Photoaffinity Labels; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence
D018685:Anesthetics, Inhalation; D001059:Apoproteins; D002250:Carbon Radioisotopes; D003574:Cytochrome c Group; D009211:Myoglobin; D010446:Peptide Fragments; D019920:Photoaffinity Labels; D011506:Proteins; C010857:apomyoglobin; D019791:Guanidine; D006221:Halothane