Abstract
Both the refolding kinetics and unfolding kinetics of ribonuclease A have been measured at the same final conditions, as a function of temperature at pH 3.9, by stopped-flow (pH-jump) experiments; absorbance changes at 240 and 286.5 nm were measured. Refolding follows first-order kinetics in the upper two-thirds of the thermal transition zone. Under the same conditions, the unfolding kinetics are biphasic; the terminal phase has the same rate constant as refolding. The biphasic kinetics of unfolding demonstrate the presence of intermediate states. Since both the refolding and unfolding kinetics are consistent with a simple sequential model, the intermediates satisfy kinetic criteria for being on the direct pathway of unfolding. At temperatures just above the transition zone, the fast phase of unfolding becomes the major kinetic phase. The rate of the slow unfolding reaction increases rapidly with temperature, and approaches the average rate of the fast phase at temperatures just above the transition zone. The entire set of kinetic results can be reproduced semiquantitatively by assignment of values to four parameters in a cooperative sequential model. However, reasons are given for the belief that this simple model will have to be generalized before it can give a realistic description of the kinetics of the unfolding reaction.
Keywords: kinetic intermediates, protein unfolding, nucleation
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