MULTIPLE KINETIC INTERMEDIATES ACCUMULATE DURING THE UNFOLDING OF HORSE CYTOCHROME-C IN THE OXIDIZED STATE

The unfolding kinetics of horse cytochrome c in the oxidized state has been studied at 10, 22, and 34 degrees C as a function of guanidine h ydrochloride (GdnHCl) concentration. Rapid (millisecond) measurements of far-UV circular dichroism (CD) as well as fluorescence quenching du e to tryptophan to heme excitation energy transfer have been used to m onitor the unfolding process. At 10 degrees C, the decrease in far-UV CD signal that accompanies unfolding occurs in two phases. The unobser vable burst phase is complete within 4 ms, while the slower phase occu rs over tens to hundreds of milliseconds. The burst phase unfolding am plitude increases cooperatively with an increase in GdnHCl concentrati on, exhibiting a transition midpoint of 3.2 M at 10 degrees C. In cont rast, no burst phase change in fluorescence occurs during unfolding at 10 degrees C. At 22 and 34 degrees C, both the fluorescence-monitored unfolding kinetics and the far-UV CD-monitored unfolding kinetics are biphasic. At both temperatures, the two probes yield burst phase unfo lding transitions that are noncoincident with respect to the transitio n midpoints as well as the dependency of the burst phase amplitudes on GdnHCl concentration. The results suggest that at least two kinetic u nfolding intermediates accumulate during unfolding. One burst phase in termediate, I-U(1), has lost virtually all the native-state secondary structure, while the other burst phase intermediate, I-U(2), has lost both secondary structure and native-like compactness. The presence of kinetic unfolding intermediates is also indicated by the nonlinear dep endence of the logarithm of the apparent unfolding rate constant on Gd nHCl concentration, which is particularly pronounced at 10 and 22 degr ees C, Analysis of the burst phase unfolding transitions obtained usin g the two probes shows that the stabilities of I-U(1) and I-U(2) decre ase steadily with an increase in temperature from 10 to 34 degrees C, suggesting that the structures present in them are stabilized principa lly by hydrogen bonding interactions.