title: Macrophage migration inhibitory factor interactions with glutathione and S-hexylglutathione.
abstract: Macrophage migration inhibitory factor (MIF) has been reported to interact with glutathione and S-hexylglutathione and to possess glutathione S-transferase activity. However, contrary to these reports, a recent NMR study concluded that MIF shows no affinity for glutathione. Re-examination of the glutathione-MIF interactions indicates that the reported increase in fluorescence upon addition of glutathione is because of pH-induced unfolding of the protein and not to any direct interactions. Circular dichroism shows that MIF remains folded from pH 4.5-7.5 but is 50% unfolded at pH 2.9 +/- 0.2. The reported increase in fluorescence can be achieved by acid titration. Under strongly buffered conditions, no fluorescence change is observed upon addition of glutathione. In contrast to the results with glutathione, MIF binds S-hexylglutathione with a Kd of 2.5 +/- 0.6 mM. Using NMR spectroscopy, a binding site which clusters around the N-terminal proline was identified. These data indicate that the binding site for S-hexylglutathione is the same as the catalytic site for the dopachrome tautomerase activity of MIF. Consequently, the binding of S-hexylglutathione as well as hexanethiol inhibits this catalytic activity.
authors.affiliation: Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510, USA.
authors.forename: M D
authors.initials: MD
authors.lastname: Swope
authors.forename: H W
authors.initials: HW
authors.lastname: Sun
authors.forename: B
authors.initials: B
authors.lastname: Klockow
authors.forename: P
authors.initials: P
authors.lastname: Blake
authors.forename: E
authors.initials: E
authors.lastname: Lolis
mesh_terms: D001665:Binding Sites / Q000502:physiology; D002942:Circular Dichroism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005453:Fluorescence; D005978:Glutathione / Q000031:analogs & derivatives* / Q000494:pharmacology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D019746:Intramolecular Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry; D008263:Macrophage Migration-Inhibitory Factors / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000494:pharmacology
chemical_list: D004791:Enzyme Inhibitors; D008263:Macrophage Migration-Inhibitory Factors; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D019746:Intramolecular Oxidoreductases; C042501:dopachrome isomerase; D005978:Glutathione; C033550:hexylglutathione