Equilibrium and transient intermediates in folding of human macrophage migration inhibitory factor

Acid, guanidinium-Cl and urea denaturations of recombinant human macrophage migration inhibitory factor (MIF) were measured using CD and fluorimetry. The acid-induced denaturation was followed by CD at 200, 222, and 278 nm an d by tryptophan fluorescence. All four probes revealed an acid-denatured st ate below pH 3 which resembled a typical molten globule. The pH transition is not two-state as the CD data at 222 nm deviated from all other probes. U rea and guanidinium-Cl denaturations (pH 7, 25 degrees C) both gave an appa rent Delta G(U)(H2O)(app) of 31 +/-3 kJ.mol(-1) when extrapolated to zero d enaturant concentration. However, denaturation transitions recorded by fluo rescence (at the same protein concentration) occurred at lower urea or guan idinium-Cl concentrations, consistent with an intermediate in the course of MIF denaturation. CD at 222 nm was not very sensitive to protein concentra tion (in 10-fold range) even though size-exclusion chromatogryphy (SEC) rev ealed a dimer-monomer dissociation prior to MIF unfolding. Refolding experi ments were performed starting from acid, guanidinium-Cl and urea-denatured states. The kinetics were multiphasic with at least two folding intermediat es. The intrinsic rate constant of the main folding phase was 5.0 +/- 0.5 s (-1) (36.6 degrees C, pH 7) and its energy of activation 155 +/- 12 kJ.mol( -1).