The thermodynamics of hydrogen isotope exchange in lysozyme: The influence of glycerol

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 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.

Viscous cosolvent effect on the ultrasonic absorption of bovine serum albumin

“…It therefore seems that the observed cosolvent effect on the UA of BSA has a dynamic, rather than thermodynamic origin. Obviously, this does not rule out cosolvent effects of thermodynamic origin as demonstrated by previous studies, using other methods (21,(25)(26)(27). However, the data presented here clearly show that the observed effect on the UA of BSA can be explained in terms of solvent viscosity.…”

“…These substances, mainly polyols, have been shown to slow down kinetic coefficients of ligand-protein interactions and subsequent biochemical processes, such as peptide hydrolysis by carboxypeptidase A (8), binding of 02 or CO to respiratory proteins (9-12), some steps in the bacteriorhodopsin cycle (13), kinetics of lactate dehydrogenase (14), ester hydrolysis by substilisin BPN (15), and the activity of cell surface phospholipase A2 (16,17), as well as membrane lipid metabolism (16, 18) and cellular secretion (19). A similar trend has been observed in the viscous cosolvents effect on hydrogen isotope exchange in lysozyme (20,21). Deviations from the viscosity dependence predicted by Eq.…”

Glycerol effects on protein flexibility: a tryptophan phosphorescence study

“…For example, Almagor et al (33) interpreting ultrasonic absorption data on serum albumin propose a model which contemplates a strong attenuation of high frequency modes of peripheral side chains accompanied by an enhancement of large amplitude, low frequency, segmental motions of the polypeptide chain. The results of H-exchange studies on lysozyme (29,30), on the other hand, show that the addition of glycerol slows down mostly the exchange of protons in rigid segments of the structure as to indicate that large amplitude fluctuations are preferentially hindered by the viscous drag. The temperature dependence of k can provide a rough indication on the kind of structural fluctuations that are principally affected by the viscous drag of the solvent.…”

“…The validity of Kramer's law has been tested experimentally for polymer dynamics (18) and by computer simulations (19,20). With proteins, increased solvent viscosity was shown to slow down rotational motions of intrinsic aromatic amino acid side-chains (21,22) and attached spinlabel probes (23) and to reduce kinetic coefficients of ligand binding (24)(25)(26)(27), of hydrogen isotope exchange (28)(29)(30), and of a number of enzymatic reaction (14,31,32). Generally, these studies have concerned rather superficial or solvent/ ligand-accessible sites of the macromolecule, which, being in more or less intimate contact with the solvent, represent rather flexible regions of the globular structure.…”

“…Within certain solvent viscosity ranges the rate is often found to obey a power law with a frictional coefficient, k, less than 1; smaller values were obtained when more internal regions of the macromolecule are concerned (23). However, a notable exception to this pattern is represented by the slow exchanging protons in lysozyme for which k is actually larger than 1 (29,30). Since slow exchange rates refer to rather tight cores of the protein structure, the large frictional coefficient found in glycerol/water solutions suggested (30) that either Gavish description of position-dependent viscosity effects is not correct or solvent viscosity is not the parameter responsible for the dramatic reduction in exchange rates.…”