Calorimetric study of G-actin denaturation

The investigation of G-actin heat denaturation at various pH of the solution by scanning microcalorimetry has shown that unfolding of G-actin is not a two-state process. Since the protein structure does not behave as a single cooperative unit during heat denaturation, it is suggested that the G-actin globule consists, at least, of two interacting domains.     

Calorimetric study of the heat and cold denaturation of beta-lactoglobulin.

Temperature-induced changes of the states of beta-lactoglobulin have been studied calorimetrically. In the presence of a high concentration of urea this protein shows not only heat but also cold denaturation. Its heat denaturation is approximated very closely by a two-state transition, while the cold denaturation deviates considerably from the two-state transition and this deviation increases as the temperature decreases. The heat effect of cold denaturation is opposite in sign to that of heat denaturation and is noticeably larger in magnitude. This difference in magnitude is caused by the temperature-dependent negative heat effect of additional binding of urea to the polypeptide chain of the protein upon its unfolding, which decreases the positive enthalpy of heat denaturation and increases the negative enthalpy of cold denaturation. The binding of urea considerably increases the partial heat capacity of the protein, especially in the denatured state. However, when corrected for the heat capacity effect of urea binding, the partial heat capacity of the denatured protein is close in magnitude to that expected for the unfolded polypeptide chain in aqueous solution without urea but only for temperatures below 10 degrees C. At higher temperatures, the heat capacity of the denatured protein is lower than that expected for the unfolded polypeptide chain. It appears that at temperatures above 10 degrees C not all the surface of the beta-lactoglobulin polypeptide chain is exposed to the solvent, even in the presence of 6 M urea; i.e., the denatured protein is not completely unfolded and unfolds only at temperatures lower than 10 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calorimetric study of the denaturation of chromatin and its components

Two-stage process of chromatin denaturation was studied. To understand the nature of heat absorption of these stages their degree of their reversibility was investigated. Some stages of heat absorption observed on the curves of chromatin and mononucleosome heat absorption reflect melting of different levels of chromatin DNA organization.     

Microcalorimetric study of the heat denaturation of carboanhydrase B

The microcalorimetric study of heat denaturation of carbonic anhydrase B has revealed that the process of denaturation of carbonic anhydrase B is accompanied by the formation of intermolecular complexes which are disrupted at a further increase of temperature. It is shown that zinc atoms stabilize the native state and do not influence the stability of intermolecular complexes.     

Calorimetric studies of the thermal denaturation of cytochrome c peroxidase

Two endotherms are observed by differential scanning calorimetry during the thermal denaturation of cytochrome c peroxidase at pH 7.0. The transition midpoint temperatures (tm) were 43.9 +/- 1.4 and 63.3 +/- 1.6 degrees C, independent of concentration. The two endotherms were observed at all pH values between 4 and 8, with the transition temperatures varying with pH. Precipitation was observed between pH 4 and 6, and only qualitative data are presented for this region. The thermal unfolding of cytochrome c peroxidase was sensitive to the presence and ligation state of the heme. Only a single endotherm was observed for the unfolding of the apoprotein, and this transition was similar to the high-temperature transition in the holoenzyme. Addition of KCN to the holoenzyme increases the midpoint of the high-temperature transition whereas the low-temperature transition was increased upon addition of KF. Binding of the natural substrate ferricytochrome c to the enzyme increases the low-temperature transition by 4.8 +/- 1.3 degrees C but has no effect on the high-temperature transition at pH 7. The presence of cytochrome c peroxidase decreases the stability of cytochrome c, and both proteins appear to unfold simultaneously. The results are discussed in terms of the two domains evident in the X-ray crystallographic structure of cytochrome c peroxidase.     

Thermodynamic investigations of proteins. II. Calorimetric study of lysozyme denaturation by guanidine hydrochloride.

The thermodynamic parameters of the denaturation of lysozyme are determined at various temperatures (25-60 degrees C) by isothermal calorimetric titrations with guanidine hydrochloride (GuHCl) and by scanning calorimetry in the presence of GuHCl. An approach for the determination of the enthalpy of preferential binding of GuHCl is proposed. It has been shown from GuHCl denaturation experiments that the net enthalpies of denaturation and the denaturational change in the heat capacity of protein can be obtained if preferential binding is taken into consideration. These results are nearly the same as in the case of thermal denaturation in the absence of denaturants. It is concluded that the states of both heat- and GuHCl-denatured lysozyme are thermodynamically indistinguishable.     

Calorimetric and circular dichroic studies of the thermal denaturation of beta-lactoglobulin

The thermal denaturation of beta-lactoglobulin in aqueous solutions at pH 5.5 and 2.0 was investigated by differential scanning calorimetry (DSC) and circular dichroic (CD) measurements. By calorimetry, the denaturation temperatures (Td), denaturation enthalpies, and specific heat capacity changes for thermal denaturation in the temperature range scanned, i.e., 20-100 degrees C. The unfolding process was found to be only partially reversible. Analysis of the far-ultraviolet CD spectra reveals that with increasing temperature the mean residue ellipticity [( theta]) becomes less negative, which reflects unfolding of the native protein. At the highest temperature of CD measurements, i.e., 80 degrees C, conformational changes are to a large extent reversible.     

Calorimetric study of the thermal denaturation of beta-lactoglobulin in the presence of urea and phosphate ions]

The denaturation of beta-lactoglobulin in solution with different content of urea and phosphates has been studied calorimetrically. It has been shown that the increase of phosphate ion concentration in solution leads to an increase of beta-lactoglobulin stability, while increase of urea concentration leads to an opposite effect. The variation of these components in solution practically does not influence the value of the heat capacity increment of beta-lactoglobulin in the considered temperature region. Accordingly the denaturation enthalpy is a linear function of temperature whose slope does not differ for solution with urea concentration less than 4.4 M. However, the absolute value of denaturation enthalpy in these solutions at corresponding temperatures differs significantly due to the heat effect of additional urea solvation during transition to the denatured state. The latter leads to a decrease of the overall denaturation enthalpy and, as a result, a shift of the enthalpy plot to higher temperatures providing conditions for studying the thermodynamic and structural characteristics of the molecule in the cold denatured-state.     

The reversible heat denaturation of chymotrypsinogen

Within a restricted range of pH and protein concentration crystalline chymotrypsinogen undergoes thermal denaturation which is wholly reversed upon cooling. At a given temperature an equilibrium exists between native and reversibly denatured protein. Within the pH range 2 to 3 the amount of denatured protein is a function of the third power of the hydrogen ion activity. The presence of small amounts of electrolyte causes aggregation of the reversibly denatured protein. A specific anion effect has been observed at pH 2 but not at pH 3. Both the reversible denaturation reaction and the reversal reaction have been found to be first order reactions with respect to protein and the kinetic and thermodynamic constants for both reactions have been approximated at pH 2 and at pH 3. Renatured chymotrypsinogen has been found to be identical with native chymotrypsinogen with respect to crystallizability, solubility, activation to δ-chymotrypsin, sedimentation rate, and behavior upon being heated. Irreversible denaturation of chymotrypsinogen has been found to depend on pH, temperature, protein concentration, and time of heating. Irreversible denaturation results in an aggregation of the denatured protein.     

A comparative thermodynamic study of heat and cold denaturation of beta-lactoglobulin]

The changes in structure and thermodynamic parameters of beta-lactoglobulin upon heat and cold denaturation have been studied using both scanning microcalorimetry and circular dichroism spectroscopy methods. It has been shown that in contrast to the heat denaturation process, the cold denaturation of beta-lactoglobulin is accompanied by an opposite heat effect. In all cases, the calorimetrically measured enthalpy of beta-lactoglobulin cold denaturation is higher than it was expected from the two-state model of denaturation transition. It has been concluded that beta-lactoglobulin cold denaturation cannot be represented by a transition between two microscopic states--native and denatured. The latter, is due to the additional process that occurs together with the disruption of the beta-lactoglobulin tertiary structure and is accompanied by increasing heat capacity. Taking into account the heat capacity contribution of this process upon calculation of the enthalpy makes it closer to the enthalpy value calculated for the two-state model of denaturation transition.     

Calorimetric and spectroscopic investigations of the thermal denaturation of wild type nitrite reductase

Nitrite reductase (NiR) is a multicopper protein, with a trimeric structure containing two types of copper site: type 1 is present in each subunit whereas type 2 is localized at the subunits interface. The paper reports on the thermal behaviour of wild type NiR from Alcaligenes faecalis S-6. The temperature-induced changes of the copper centres are characterized by optical spectroscopy and electron paramagnetic resonance spectroscopy, and by establishing the thermal stability by differential scanning calorimetry. The calorimetric profile of the enzyme shows a single endothermic peak with maximum heat absorption at T_m ≈ 100 °C, revealing an exceptional thermal stability. The thermal transition is irreversible and the scan rate dependence of the calorimetric trace indicates that the denaturation of NiR is kinetically controlled. The divergence of the activation energy values determined by different methods is used as a criterion for the inapplicability of the one-step irreversible model. The best fit of the DSC profiles is obtained when the classical Lumry–Eyring model, N ? U ? F, is considered. The simulation results indicate that the irreversible step prevails on the reversible one. Moreover, it is found that the conformational changes within the type-1 copper environments precede the denaturation of the whole protein. No evidence of protein dissociation within the temperature range investigated was observed.     

Changes in heat capacity upon protein denaturation

By the use of infrared spectroscopy it has been shown that the configurational heat capacity of hydrogen bonds represents the principal contribution into the denaturational change of heat capacity of proteins.