Kinetic evidence for intermediate states in the unfolding of ribonuclease A. II. Kinetics of exposure to solvent of a specific dinitrophenyl group

Recent kinetic studies of the reversible unfolding of ribonuclease A at low pH indicate that intermediate states are present (Tsong, Baldwin &; Elson, 1971). The intermediates are detected by fast reactions in the millisecond time-range which accompany the slow unfolding; both reactions are measured at 286.5 nm by exposure of buried tyrosine groups. To find out whether the fast and slow processes are separate unfolding reactions that involve different tyrosine groups, or whether they are different kinetic phases of a general unfolding reaction, we have studied 41-dinitrophenyl ribonuclease A (Ettinger &; Hirs, 1968). The kinetics of exposure of the single dinitrophenyl group, measured at 358 nm, are biphasic, and the rates of both the fast and slow processes agree closely with those measured by tyrosine groups at 286.5 nm. The results are consistent with the postulate that the slow process is an almost steady-state unfolding across a sequence of steps and the fast process is a transient phase involving individual steps in unfolding.