DSC study of reversible and irreversible thermal denaturation of concentrated globular protein solutions

A calorimetric study of the thermal denaturation of bovine serum albumin, RNAase and catalase in concentrated solutions (crystals) has been carried out. The results obtained for RNAase studied within the pH range 2.5-8.5 show that for concentrated solutions there is an interval of pH where, on cooling of the solution which had undergone denaturation, its renaturation is observed. In the case of concentrated and dilute solutions of RNAase these intervals coincide. The study of RNAase under such conditions at various heating rates shows that there is a range of rates in which the process of denaturation of concentrated solutions can be considered as reversible. The dependences of Td and Hd on pH and concentration of solutions have been determined. The denaturation enthalpy of concentrated solutions like in dilute ones, has been found to be independent of the pH of solutions, and the experimentally registered change has been proved to be the result of its dependence on temperature. A new method of determination of protein denaturation enthalpy under the conditions of intensive molecule aggregation is suggested. The forms of irreversibility as appearing in the calorimetric experiment were determined by comparing reversible and irreversible denaturation under continuous and step-heating regimes. It is shown that the decrease in Tmax and the narrowing of the heat absorption peak in the case of decreasing heating rates of protein solutions, observed under certain environmental conditions, results from the irreversibility of the denaturation process.     

Equilibrium and kinetic studies on reversible and irreversible denaturation of micrococcal nuclease

The effect of pH and temperature on the thermal denaturation of micrococcal nuclease wer4e investigated. The ranges employed were between pH3.30 and pH9.70 and between 10 degrees C and 85 degrees C, respectively. The reversible denaturation involved in the whole process was clearly discriminated from the irreversible one. The former took place with a large enthalpy change of 384 kJ mol(-1) at pH 9.70, where the enzyme exhibited it s maximum activity. The latter probably led to aggregation because the successive long incubation after complete deactivation caused precipitation. A reasonable scheme explaining the process involving both denaturations was proposed and the kinetic on the irreversible deactivation was performed. It was revealed that the irreversible deactivation involved two types of reactions whose activation energies were relatively small: 22.2 kJ mol(-1) and 18.8kJ mol(-1). The presence of sucrose suppressed the reversible denaturation without significant influence on enthalpy change, whereas it affected little the irreversible deactivation kinetically. The effects of pH change and addition of sucrose on the denaturation were discussed thermodynamically, especially in terms of the entropy change. (c) 1994 John Wiley & Sons, Inc.     

alpha-B- and alpha-A-crystallin prevent irreversible acidification-induced protein denaturation

alpha-Crystallin (alpha), a major structural protein of the mammalian lens, is a large, physically heterogeneous macromolecule with an average molecular weight of approximately 800 kDa and is composed of two 20-kDa polypeptides designated as alphaA and alphaB. A line of evidence strongly suggests that alphaB may have an essential nonlenticular function. Here it is demonstrated that alphaB can bind partially denatured enzymes effectively at acidic pH and prevent their irreversible aggregation, but cannot prevent loss of enzyme activity. However, when the inactive luciferase bound to alphaB was treated with reticulocyte lysate (a rich source of molecular chaperones) and an ATP-generating system, more than 50% of the original luciferase activity could be recovered. Somewhat less activation was observed when alphaA-bound enzyme or the alpha-bound enzyme was renatured similarly. The overall results suggest that alpha acts as a chaperone to stabilize denaturing proteins at acidic pH so that at a later time they can be reactivated by other chaperones.     

Influence of transition rates and scan rate on kinetic simulations of differential scanning calorimetry profiles of reversible and irreversible protein denaturation.

The thermodynamic parameters characterizing protein folding can be obtained directly using differential scanning calorimetry (DSC). They are meaningful only for reversible unfolding at equilibrium, which holds for small globular proteins; however, the unfolding or denaturation of most large, multidomain or multisubunit proteins is either partially or totally irreversible. The simplest kinetic model describing partially irreversible denaturation requires three states: Formula [see text] We obtain numerical solutions for N, U, and D as a function of temperature for this model and derive profiles of excess specific heat (Cp) in terms of the reduced variables v/ki and k1/k3, where v is the scan rate. The three-state model reduces to the two-state reversible or irreversible models for very large or very small values of k1/k3, respectively. The apparent transition temperature (Tapp) is always reduced by the irreversible step (U-->D). For all values of k3, Tapp is independent of v/k1 at sufficiently slow scan rates, even when denaturation is highly irreversible, but increases identically for all models at fast scan rates in which case the excess specific heat profile is determined by the rate of unfolding. Accurate values of delta H and delta S can be obtained for the reversible step only when k1 is more than 2000-50,000 times greater than k3. In principle, approximate values for the ratio k1/k3 can be obtained from plots of fraction unfolded vs fraction irreversibly denatured as a function of temperature; however, the fraction irreversibly denatured is difficult to measure accurately by DSC alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modeling of irreversible thermal protein denaturation at varying temperature. II. The complete kinetic model of Lumry and Eyring.

The model for thermal denaturation of proteins involving consecutive reversible and irreversible steps (Lumry and Eyring model) has been analyzed. The most general case, when equilibrium in the first step is established slowly in comparison with the rate of the second step and the heat effect value for the second step is either greater than or less than zero, has been considered. The theoretical dependences of excess heat capacity on temperature have been constructed. The variation of the shape of the theoretical curves with varied values of the enthalpy change for the second step, Arrhenius equation parameters for both steps, and the scanning rate has been studied.     

Analysis of kinetic data for irreversible enzyme inhibition.

Many organophosphorus compounds are irreversible inhibitors of acetylcholinesterase. The methods used in the literature to determine the inhibition kinetic constants usually involve either manual determination of the slope at various points along the inhibition progress curve or fitting polynomials to the curve. The present study investigates the use of non-linear-regression analysis to determine the various parameters. A method is suggested that yields accurate values for the inhibition constants under a range of circumstances.     

Interpretation of thermograms of protein denaturation in non-equilibrium conditions

Data are presented concerning the effect of heating rate on the denaturation parameters of small and oligomeric globular proteins: Kunitz trypsin inhibitor from soybeans and 1,5-Ribulose Bisphosphate Carboxylase from tobacco leaves. Substantional dependence of denaturation temperature on the heating rate reflects non-equilibrium pattern of denaturation of these proteins under experimental conditions. To interpret these data a kinetic approach is proposed, which permits determination of equilibrium value of the denaturation temperature and of the constant of de- and renaturation rate. The conformation transitions in the proteins studied are shown to be relatively slow processes. Their rate is comparable to the velocity of temperature change in a calorimeter, which is the cause of non-equilibrium effects in a calorimetric experiment.     

Analysis of differential scanning calorimetry data for proteins. Criteria of validity of one-step mechanism of irreversible protein denaturation

We consider in this work the analysis of the excess heat capacity C(p)(ex) versus temperature profiles in terms of a model of thermal protein denaturation involving one irreversible step. It is shown that the dependences of ln C(p)(ex) on 1 T (T is the absolute temperature) obtained at various temperature scanning rates have the same form. Several new methods for estimation of parameters of the Arrhenius equation are explored. These new methods are based on the fitting of theoretical equations to the experimental heat capacity data, as well as on the analysis of the dependence d(ln C (p)(ex)) d ( 1 T ) on 1 T . We have applied the proposed methods to calorimetric data corresponding to the irreversible thermal denaturation of Torpedo californica acetylcholinesterase, cellulase from Streptomyces halstedii JM8, and lentil lectin. Criteria of validity for the one-step irreversible denaturation model are discussed.     

Synergistic inhibition of acid-induced protein denaturation by trehalose and NaCl: Thermodynamic and kinetic studies

It is known that trehalose and sodium chloride (NaCl) can both effectively inhibit acid-induced protein denaturation, but the thermodynamic and kinetic behaviors of acid-induced protein unfolding synergistically inhibited by trehalose and NaCl are unclear. In this study, the synergistic inhibition effects of trehalose and NaCl on the acid-induced unfolding of ferricytochrome c were studied at pH 2.0. Thermodynamic parameters were firstly derived based on fluorescence spectroscopic data. Then, kinetic behaviors were studied using stopped-flow fluorescence spectroscopy. It was found that the kinetics of the acid-induced protein unfolding transformed from a triphasic process (i.e., fast, intermediate and slow phases) into a biphasic one (i.e., intermediate and slow phases) and then a single slow phase process with increasing either trehalose or NaCl concentration in the mixture. The rate constants for all the unfolding phases change slightly, while the amplitudes for the fast and intermediate phases diminish greatly with increasing the concentration of trehalose or NaCl. This clearly indicates that the mixture of trehalose and NaCl could synergistically inhibit acid-induced protein unfolding by reducing the extent of protein conformational changes, thus inducing a stable molten-globule state at higher concentrations of the agents.     

Absence of kinetic thermal stabilization in a hyperthermophile rubredoxin indicated by 40 microsecond folding in the presence of irreversible denaturation

The striking kinetic stability of many proteins derived from hyperthermophilic organisms has led to the proposal that such stability may result from a heightened activation barrier for unfolding independent of a corresponding increase in the thermodynamic stability. This in turn implies a corresponding retardation of the folding reaction. A commonly cited model for kinetic thermal stabilization is the rubredoxin from Pyrococcus furiosus (Pf), which exhibits an irreversible denaturation lifetime at 100 degrees C of nearly a week. Utilizing protein resonances shifted well outside of the random coil chemical shift envelope, nuclear magnetic resonance (NMR) chemical exchange measurements on Pf rubredoxin as well as on the mesophile Clostridium pasteurianum (Cp) rubredoxin demonstrate reversible thermal transition temperatures of 144 degrees C (137 degrees C for the N-terminal modified A2K variant) and 104 degrees C, respectively, with similar (un)folding rates of approximately 25,000 s(-1), only modestly slower than the diffusion controlled rate. The absence of a substantial activation barrier to rubredoxin folding as well as the similar folding kinetics of the mesophile protein indicate that kinetic stabilization has not been utilized by the hyperthermophile rubredoxin in achieving its extreme thermal stability. The two-state folding kinetics observed for Pf rubredoxin contradict a previous assertion of multiphasic folding based on hydrogen exchange data extrapolated to an estimated midpoint of transition temperature (T(m)) of nearly 200 degrees C. This discrepancy is resolved by the observation that the base-catalyzed hydrogen exchange of the model dipeptide (N-acetyl-L-cysteine-N-methylamide)4-Cd2+ is 23-fold slower than that of the free cysteine model dipeptide used to normalize the Pf rubredoxin hydrogen exchange data.     

Thermodynamic and kinetic study of alpha-lactalbumin denaturation

The measured curves of alpha-lactalbumin denaturation kinetics in the presence of urea are described in terms of a single phase. Its rate constant does not depend on the parameter detected. Both kinetic and equilibrium measurements show an accumulation of a protein molecule state in the denaturation region which differs from the native one in its fluorescence properties.     

Cold denaturation and heat denaturation of Streptomyces subtilisin inhibitor. 1. CD and DSC studies

Cold denaturation and heat denaturation of the protein Streptomyces subtilisin inhibitor (SSI) were studied in the pH range 1.84-3.21 and in the temperature range -3-70 degrees C by circular dichroism and scanning microcalorimetry. The native structure of the protein was apparently most stabilized at about 20 degrees C and was denatured upon heating and cooling from this temperature. Each denaturation was reversible and cooperative, proceeding in two-state transitions, that is, from the native state to the cold-denatured state or from the native state to the heat-denatured state. The two denatured states, however, were not perfect random-coiled structures, and they differed from each other, indicating that there exist three states in this temperature range, i.e., cold denatured, native, and heat denatured. The difference between the cold and heat denaturations was indicated first by circular dichroism. The isodichroic point for the transition from the native state to the cold-denatured state was different from that from the native state to the heat-denatured state in the pH range between 3.21 and 2.45. Moreover, molar ellipticity for the cold-denatured state was different from that of the heat-denatured state, and the transition from the former to the latter was observed at pH values below 2. Values of van't Hoff enthalpies from the native state to the heat-denatured state at pH values between 3.21 and 2.45 were obtained by curve fitting of the CD data, and delta Cp = 1.82 (+/- 0.11) [kcal/(mol.K)] was obtained from the linear plot of the enthalpies against temperature. The parameters obtained from the heat denaturation studies gave curves for delta G zero which were not in agreement with the experimental data in the cold denaturation region when extrapolated to the low temperature. Moreover, the value of the apparent delta Cp for the cold denaturation in the pH range 3.03-2.45 was estimated to be different from that for the heat denaturation, indicating that the mechanism of the cold denaturation of SSI is different from a simple cold denaturation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetic study on the irreversible thermal denaturation of yeast phosphoglycerate kinase

Differential scanning calorimetry transitions for the irreversible thermal denaturation of yeast phosphoglycerate kinase at pH 7.0 are strongly scanning-rate dependent, suggesting that the denaturation is, at least in part, under kinetic control. To test this possibility, we have carried out a kinetic study on the thermal inactivation of the enzyme. The inactivation kinetics are comparatively fast within the temperature range of the calorimetric transitions and can be described phenomenologically by the equation dC/dt = -alpha C2/(beta + C), where C is the concentration of active enzyme at a given time, t, and alpha and beta are rate coefficients that depend on temperature. This equation, together with the values of alpha and beta (within the temperature range 50-59 degrees C) have allowed us to calculate the fraction of irreversibly denatured protein versus temperature profiles corresponding to the calorimetric experiments. We have found that (a) irreversible denaturation takes place during the time the protein spends in the transition region and (b) there is an excellent correlation between the temperatures of the maximum of the calorimetric transitions (Tm) and the temperatures (Th) at which half of the protein is irreversibly denatured. These results show that the differential scanning calorimetry transitions for the denaturation of phosphoglycerate kinase are highly distorted by the rate-limited irreversible process. Finally, some comments are made as to the use of equilibrium thermodynamics in the analysis of irreversible protein denaturation.     

Kinetic study into the irreversible thermal denaturation of bacteriorhodopsin

We report on a differential scanning calorimetry study of native purple membranes under the following solvent conditions: 50 mM carbonate-bicarbonate, 100 mM NaCl, pH 9.5 and 190 mM phosphate, pH 7.5. The calorimetric transitions for bacteriorhodopsin denaturation are highly scanning-rate dependent, which indicates that the thermal denaturation is under kinetic control. This result is confirmed by a spectrophotometric study on the kinetics of the thermal denaturation of this protein. The calorimetric data at pH 9.5 conform to the two-state irreversible model. Comments are made regarding the information obtainable from differential scanning calorimetry studies on bacteriorhodopsin denaturation and the effect of irreversibility on the stability of membrane proteins. Correspondence to: J. M. Sanchez-Ruiz     

Interpretation of 51Cr-release data: a kinetic analysis.

An analysis of 51Cr-release data from cell-mediated cytotoxicity assays, plotted in the form of effector cell dilution curves, is analyzed in detail in terms of a saturation kinetics model for effector-target interaction. The effect of nonimmune (bystander) cells in the effector population is particularly examined. The theoretical development of the saturation model predicts that, at low concentrations of target cells, increasing proportions of bystander cells should broaden the range of effector cell concentration required to achieve a given increment of target cell lysis, but that at high target cell concentration this effect should disappear. Experimental validation of this prediction is presented.     

Two-state irreversible thermal denaturation of muscle creatine kinase.

Thermal denaturation of creatine kinase from rabbit skeletal muscle has been studied by differential scanning calorimetry. The excess heat capacity vs. temperature profiles were independent of protein concentration, but strongly temperature scanning rate-dependent. It has been shown that thermal denaturation of creatine kinase satisfies the previously proposed validity criteria for the two-state irreversible model [Kurganov et al., Biophys. Chem.70 (1997) 125]. The energy activation value has been calculated to be 461.0 +/- 0.7 kJ/mol.     

Dissociative mechanism for irreversible thermal denaturation of oligomeric proteins

Protein stability is a fundamental characteristic essential for understanding conformational transformations of the proteins in the cell. When using protein preparations in biotechnology and biomedicine, the problem of protein stability is of great importance. The kinetics of denaturation of oligomeric proteins may have characteristic properties determined by the quaternary structure. The kinetic schemes of denaturation can include the multiple stages of conformational transitions in the protein oligomer and stages of reversible dissociation of the oligomer. In this case, the shape of the kinetic curve of denaturation or the shape of the melting curve registered by differential scanning calorimetry can vary with varying the protein concentration. The experimental data illustrating dissociative mechanism for irreversible thermal denaturation of oligomeric proteins have been summarized in the present review. The use of test systems based on thermal aggregation of oligomeric proteins for screening of agents possessing anti-aggregation activity is discussed.