The temperature dependence of the hydrogen-tritium and deuterium-hydrogen exchange reactions in poly(DL-alanine) has been reexamined. The results indicate a significant contribution to the observed exchange rates from the water-catalyzed reaction at pD values near pDmin. The activation enthalpy for water-catalyzed deuterium-hydrogen exchange in poly(DL-alanine) is found to be 21 kcal mol-1. As a result, the contribution to the observed exchange rate from the water-catalyzed reaction increases with increasing temperature which in turn leads to broad, shallow pD minima and the appearance of apparent reaction orders with respect to [D+] and [OD-] that are substantially less than first order over an extended range of pD values. The importance of water catalysis in protein hydrogen exchange is demonstrated by a reanalysis of data for the exchange of single protons in bovine pancreatic trypsin inhibitor [Hilton, B.D., & Woodward, C. K. (1979) Biochemistry 18, 5834; Richarz, R., Sehr, P., Wagner, G., & Wüthrich, K. (1979) J. Mol. Biol. 130, 19]. The pD dependence of these protons can be explained in terms of an increased contribution from water catalysis.

A new method is described that makes use of the empirical enthalpy--entropy compensation behavior of a related series of processes for deriving the activation enthalpy and entropy probability density functions from the corresponding rate constant density function. The method has been applied to data obtained from a study of the temperature dependence of hydrogen-tritium exchange in lysozyme. Analysis of the temperature dependence of tj, the time required to reach a particular number of hydrogens remaining unexchanged, provides estimates of delta G#, delta H#, and delta S# for the exchange process. The results are consistent with the notion of two mechanisms of exchange characterized by different activation energies. Increases in delta H# are compensated by corresponding increases in delta S#. The compensation plot, however, reveals two distinct apparent compensation temperatures, which reflect the operation of two qualitatively different mechanisms of exchange. The faster hydrogens exchange with delta H# values between 8 and 18 kcal X mol-1 and are characterized by a high compensation temperature of 470 K. The slower hydrogens exchange with delta H# values that reach 40 kcal X mol-1 and display a compensation temperature of congruent to 360 K. The latter is associated with a thermal unfolding mechanism of exchange.

The temperature dependence of hydrogen isotope exchange rates for lysozyme in 5 molal aqueous glycerol and for poly(D,L-alanine) in a range of glycerol concentrations from 0 molal to 8 molal have been determined. The activation enthalpy of base-catalyzed exchange for poly(D,L-alanine) in water is 4 kcallmol and passes through a minimum a t about 2 molal glycerol before returning to a value of 4 kcallmol a t 4 molal glycerol. Exchange rates for lysozyme have been analyzed with transition state and Kramers's theories. The activation parameters for exchange of protons in lysozyme in the presence of 5 molal glycerol show a similar qualitative behavior to those determined for exchange in the absence of glycerol [ R. B. Gregory et al. ( 1982) Biochemistry 24, 6523-65301. The activation enthalpies and entropies for the fast-exchanging protons show a gentle increase as H ( t ) , the number of hydrogens remaining unexchanged, decreases. By contrast, the activation parameters for the slowest exchanging protons [ H ( t ) < 201 increase dramatically as H ( t ) decreases. As in water, the activation parameters for exchange of the fast-and slow-exchanging protons in glycerol solution are characterized by two distinct compensation temperatures (510 k 100 K for the fast protons and 340 f 40K for the slow protons). These values are not significantly different from those determined for exchange in water.The activation parameters, A H t and T A S f , are both several kcal/mol lower for exchange in glycerol solutions compared with water. Enthalpy and entropy changes for the thermal unfolding of lysozyme in glycerol solutions [results of K. Gekko ( 1982) J. Biochem. 19, 1197-12041 are larger than those determined for unfolding in water. This suggests that exchange does not involve local unfolding of segments of the protein. There is evidence for a contribution from thermal unfolding for exchange of the slowest protons in glycerol solutions which is not observed in water.Analysis of exchange rates for the fast protons as a function of temperature and viscosity with Kramers' equation provides values of the viscosity exponent. This is found to increase with decreasing values of H ( t ) . These results are not easily rationalized with Gavish's model [ (1980) Phys. Reu. Lett. 44, 11601 of a "position-dependent, internal protein viscosity" and suggest that the Gavish model of the friction coefficient is incorrect. A dissection of the effect of glycerol on exchange of the fast protons into viscosity and thermodynamic contributions is not possible with the present data. Both factors are suggested to influence exchange of the fast protons.