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The unfolding transition and kinetic refolding of dimeric creatine kinase after urea denaturation were monitored by intrinsic fluorescence and far ultraviolet circular dichroism. An equilibrium intermediate and a kinetic folding intermediate were identified and characterized. The fluorescence intensity of the equilibrium intermediate is close to that of the unfolded state, whereas its ellipticity at 222 nm is about 50% of the native state. The transition curves measured by these two methods are therefore non-coincident. The kinetic folding intermediate, formed during the burst phase of refolding under native-like conditions, possesses 75% of the native secondary structure, but is mostly lacking in native tertiary structure. In moderate concentrations of urea, only the initial, rapid change in fluorescence intensity or negative ellipticity is observed, and the final state values do not reach the equivalent unfolding values. The unfolding and refolding transition curves measured under identical conditions are non-coincident within the transition from intermediate to fully unfolded state. It is observed by SDS-PAGE that disulfide bond-linked dimeric or oligomeric intermediates are formed in moderate urea concentrations, especially in the refolding reaction. These rapidly formed, soluble intermediates represent an off-pathway event that leads to the hysteresis in the refolding transition curves.

The activation and inactivation of dihydrofolate reductase from chicken liver during denaturation in a wide concentration range of urea are compared with changes in intrinsic fluorescence. At 2 M urea the enzyme is activated 3.6-fold and is stable up to 12 h in the activated form. At 4 M urea, the enzyme activity increases about 5-fold initially but the activated enzyme loses activity rapidly to a level well below that of the native enzyme. The activated enzyme is stabilized in presence of either DHF or NADPH. The Kd and Km of the enzyme for the substrates at various urea concentrations were determined and compared. In the presence of 3 M urea, the values of Kd for DHF and NADPH increase 4-fold and 10-fold, respectively, whereas the corresponding Km values increase 25-fold and 3-fold. A large increase in Vmax is mainly responsible for the activation. The inactivation and unfolding in urea are both biphasic processes. For the fast phase, the rate constant of inactivation is 10-fold greater than that of unfolding in 4 M urea. The effect of (NH4)2SO4 on the activation and unfolding of the enzyme was also studied. The results suggest that the active site of the enzyme is more easily perturbed by denaturants; and the activated enzyme appears to have a more open and flexible conformation at the active site, which is favorable for the full expression of the catalytic power of the enzyme. A scheme for the sequential activation and inactivation of DHFR accompanying its unfolding by increasing concentrations of urea is proposed.