KINETICS OF HYDROGEN-BOND BREAKAGE IN THE PROCESS OF UNFOLDING OF RIBONUCLEASE-A MEASURED BY PULSED HYDROGEN-EXCHANGE

A sensitive test for kinetic unfolding intermediates in ribonuclease A (EC 3.1.27.5) is performed under conditions where the enzyme unfolds slowly (10 degrees C, pH 8.0, 4.5 M guanidinium chloride). Exchange of peptide NH protons (H-2-H-1) is used to monitor structural opening of individual hydrogen bonds during unfolding, and kinetic models are de veloped for hydrogen exchange during the process of protein unfolding. The analysis indicates that the kinetic process of unfolding can be m onitored by EX1 exchange (limited by the rate of opening) for ribonucl ease A in these conditions. Of the 49 protons whose unfolding/exchange kinetics was measured, 47 have known hydrogen bond acceptor groups. T o test whether exchange during unfolding follows the EX2 (base-catalyz ed) or the EX1 (uncatalyzed) mechanism, unfolding/exchange was measure d both at pH 8.,O and at pH 9.0, A few faster-exchanging protons were found that undergo exchange by both EX1 and EX2 processes, but the 43 slower-exchanging protons at pH 8 undergo exchange only by the EX1 mec hanism, and they have closely similar rates. Thus, it is likely that a ll 49 protons undergo EX1 exchange at the same rate. The results indic ate that a single rate-limiting step in unfolding breaks the entire ne twork of peptide hydrogen bonds and causes the overall unfolding of ri bonuclease A. The additional exchange observed for some protons that f ollows the EX2 mechanism probably results from equilibrium unfolding i ntermediates and will be discussed elsewhere.