Abstract
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 kcal/mol and passes through a minimum at about 2 molal glycerol before returning to a value of 4 kcal/mol at 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, 6523–6530]. 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) < 20] 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 ± 100 K for the fast protons and 340 ± 40K for the slow protons). These values are not significantly different from those determined for exchange in water.
The activation parameters, Δ__H__^‡^, and T__Δ__S^‡^, 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, 1197–1204] 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. Rev. Lett. 44, 1160] 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.