Bovine serum albumin in aqueous guanidine hydrochloride solutions. Preferential and absolute interactions and comparison with other systems

Reisler, Emil; Haik, Yeheskiel; Eisenberg, Henryk

doi:10.1021/bi00621a006

Cited by 105 publications

(58 citation statements)

References 29 publications

(39 reference statements)

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“…Because all sites must be in contact with either water or cosolvent molecules, is it possible to deduce their numbers from equilibrium binding measurements? A relation has been derived by various intuitive approaches (7,17,(27)(28)(29):…”

Section: Site Occupancymentioning

confidence: 99%

Protein-solvent preferential interactions, protein hydration, and the modulation of biochemical reactions by solvent components

Timasheff

2002

Proc. Natl. Acad. Sci. U.S.A.

627

663

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Solvent additives (cosolvents, osmolytes) modulate biochemical reactions if, during the course of the reaction, there is a change in preferential interactions of solvent components with the reacting system. Preferential interactions can be expressed in terms of preferential binding of the cosolvent or its preferential exclusion (preferential hydration). The driving force is the perturbation by the protein of the chemical potential of the cosolvent. It is shown that the measured change of the amount of water in contact with protein during the course of the reaction modulated by an osmolyte is a change in preferential hydration that is strictly a measure of the cosolvent chemical potential perturbation by the protein in the ternary water-protein-cosolvent system. It is not equal to the change in water of hydration, because water of hydration is a reflection strictly of protein-water forces in a binary system. There is no direct relation between water of preferential hydration and water of hydration. For the better part of a century, it has been common practice to modulate biochemical (biological) reactions by the addition to the aqueous solvent (dilute buffer) of compounds that were required at high concentration (ϳ0.5 M or higher) to exert their effect. For example, sucrose and glycerol were used to stabilize biological systems, whereas urea and guanidine hydrochloride were used to solubilize coagulated systems and to unfold (denature) proteins. The aim in the use of these additives, referred to as cosolvents, was to displace to the right or left the chemical equilibrium, Reactant º Product. Although this practice was widespread, a theoretical underpinning was lacking until the discovery by Wyman in 1948 of the phenomenon of linked functions (1) and his development of the linkage relationship equations (2, 3), which showed the necessary thermodynamic interdependence between the displacement of a chemical equilibrium and the change in binding of a ligand to the system during the course of the reaction. Complete understanding of the action of cosolvents in such modulation required the combination of the Wyman linkage relations with the multicomponent solution thermodynamics theory developed by Kirkwood and Goldberg (4), Stockmayer (5), and Scatchard (6).The basic Wyman linkage equation states that, at any ligand concentration, m L , the gradient of the equilibrium constant with respect to ligand activity is equal to the change in the binding of the ligand to the biological system during the course of the reaction (at constant temperature and pressure that will be maintained throughout):where K is the equilibrium constant of the reaction, a L is the activity of the ligand (a L ϭ m L ␥ L ), L Prod and L React are the bindings of the ligand to the two end states of the reaction, and m L and ␥ L are the molal concentration and activity coefficient of the ligand. [In our notation, the subscripts W, L, and P refer to water, ligand (cosolvent), and protein (macromolecule), respectively.] Tanford, in 1969 (7), showed that ...

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Section: Site Occupancymentioning

confidence: 99%

Protein-solvent preferential interactions, protein hydration, and the modulation of biochemical reactions by solvent components

Timasheff

2002

Proc. Natl. Acad. Sci. U.S.A.

627

663

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“…The dashed curve demonstrates that the values of the constants obtained from the expansion formalism [Eqs. (12) and (13)] are accurate, especially at low to moderate concentrations.…”

Section: Site Occupationmentioning

confidence: 99%

The thermodynamics of solvent exchange

1994

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A model for solvation in mixed solvents, which was developed for the free energy and preferential interaction [J. A. Schellman (1987), Biopolymers, Vol. 26, pp. 549-559; (1990), Biophysical Chemistry, Vol. 37, pp. 121-140; (1993), Biophysical Chemistry, Vol. 45, pp. 273-279], is extended in this paper to cover the thermal properties: enthalpy, entropy, and heat capacity. An important result is that the enthalpy of solvation H(ex)2 responds directly to the fraction of site occupation. This differs from the free energy G(ex)2 and preferential interaction gamma 32, which are measures of the excess binding above a random distribution of solvent molecules. In other words, the enthalpy is governed by K while G(ex)2 and gamma 32 are governed by (K-1) where K is the equilibrium constant on a mole fraction scale [Schellman (1987)]. The solvation heat capacity Cpex2 consists of two term: (1) the intrinsic heat capacity of species in solution with no change in composition, and (2) a term that accounts for the change in composition that accompanies solvent exchange. Binding to biological macromolecules is heterogeneous but experimentalists must use binding isotherms that assume the homogeneity of sites. Equations are developed for the interpretation of the experimental parameters (number of sites nexp, equilibrium constant Kexp, and enthalpy, delta hexp), when homogeneous formulas are applied to the heterogeneous case. It is shown that the experimental parameters for the occupation and enthalpy are simple functions of the moments of the distribution of equilibrium constants over the sites. In general, nexp is greater than the true number of sites and Kexp is greater than the average of the equilibrium constants. The free energy and preferential interaction can be fit to a homogeneous formula, but the parameters of the curve are not easily represented in terms of the moments of distributions over the sites. The strengths and deficiencies of this type of thermodynamic model are discussed.

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“…The kinetic constants of inactivation k (k = In 2/t1, 2) are plotted for the various salt concentrations in Fig. 2.…”

Section: Temperature and Salt Concentration Dependence Of Mulute Dehymentioning

confidence: 99%

“…Concepts relating to 'binding' of low-molecular-weight components to large particles in solution are largely operational and different methods may yield different results. We have used an approach based on volume exclusion, as manifested by density increments at specified compositions and thermodynamic potentials of water and salt [2,3]. This concept has previously yielded reasonable values for hydration and interaction with salts of DNA and of non-halophilic proteins [l].…”

mentioning

confidence: 99%

Structure and Activity of Malate Dehydrogenase from the Extreme Halophilic Bacteria of the Dead Sea. 2. Inactivation, Dissociation and Unfolding at NaCl Concentrations below 2 M. Salt, Salt Concentration and Temperature Dependence of Enzyme Stability

1981

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The stability of halophilic malate dehydrogenase increases with increasing salt concentration and with decrease in temperature. Stabilization by various salts, at high salt concentrations, follows the Hofmeister series. The enzyme inactivation rates closely match dissociation of the dimeric enzymes into monomeric subunits and unfolding of the polypeptide chains, as followed by velocity sedimentation, light. scattering and circular dichroism measurements. The a-helix content goes to zero upon denaturation. Unusual water and salt binding properties of the native enzyme (cf. preceding paper, in this journal) are believed to be largely lost upon enzyme dissociation and unfolding. These properties thus seem to be associated with the intact structure of the enzyme.In our previous work [l] we have shown that, although malate dehydrogenase (h) becomes unstable at NaCl concentrations lower than about 2.5 M, enzyme activity and structure is maintained for short times at NaCl concentrations as low as 1 M. It was therefore possible to provide a description of the properties of the active enzyme over the NaCl concentration range 1-5 M, by performing a series of diffusion, sedimentation and circular dichroism experiments. These essentially could be considered as being performed at 'zero time', before any significant changes in enzymatic activity or conformation arises.The recent striking observation resulting from the above work is the fact that malate dehydrogenase (h), as well as glutamate dehydrogenase (h), complex in striking fashion with large amounts of both water and salt. Concepts relating to 'binding' of low-molecular-weight components to large particles in solution are largely operational and different methods may yield different results. We have used an approach based on volume exclusion, as manifested by density increments at specified compositions and thermodynamic potentials of water and salt [2,3]. This concept has previously yielded reasonable values for hydration and interaction with salts of DNA and of non-halophilic proteins [l]. The behavior of the halophilic enzymes is distinctly different from that of the non-halophilic macromolecules examined. We shall present evidence in the present work to suggest that these unusual hydration and salt binding characteristics are largely lost when malate dehydrogenase (h) dissociates and unfolds. An indication is thus provided for a mechanism of adaptation to extreme conditions of high salt concentration which is dependent on the intactness of the active enzyme structure.Ahbreviaiions. Malate dehydrogenase (h), halophilic malate dchydrogenase; glutamate dehydrogenase (h), halophilic glutamate dehydrogenase.Enzymes. Malate dehydrogenase (EC 1.1.1.37); glutamate dehydrogenase [NAD(P)+] (EC 1.4.1.3).In an earlier investigation Mevarech and Neumann [4] described the reversible inactivation of malate dehydrogenase (h) at low NaCl concentration. They suggested that inactivation of the dimeric enzyme involved dissociation into two subunits, since the kinetics of reactivation...

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Bovine serum albumin in aqueous guanidine hydrochloride solutions. Preferential and absolute interactions and comparison with other systems

Cited by 105 publications

References 29 publications

Protein-solvent preferential interactions, protein hydration, and the modulation of biochemical reactions by solvent components

Protein-solvent preferential interactions, protein hydration, and the modulation of biochemical reactions by solvent components

The thermodynamics of solvent exchange

Structure and Activity of Malate Dehydrogenase from the Extreme Halophilic Bacteria of the Dead Sea. 2. Inactivation, Dissociation and Unfolding at NaCl Concentrations below 2 M. Salt, Salt Concentration and Temperature Dependence of Enzyme Stability

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