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)     

Heat and cold denaturation of beta-lactoglobulin B.

The thermal denaturation of bovine beta-lactoglobulin B was investigated by high-sensitivity differential scanning microcalorimetry between pH 1.5 and 3.0 in 20 mM phosphate buffer. The process was found to be a reversible, two-state transition. Progressive addition of guanidine hydrochloride at pH 3.0 leads to the appearance of a low-temperature calorimetric endotherm, corresponding to the cold renaturation of the protein. Circular dichroism experiments have confirmed the low and high temperature denaturation processes, and have shown some structural differences between both denatured states of beta-lactoglobulin B.     

Hydration and thermal denaturation of beta-lactoglobulin. A calorimetric study.

The thermal properties of the beta-lactoglobulin-water system were investigated by differential scanning calorimetry in the temperature range from -50 to 130 degrees C. Determination of the heat and temperature of fusion of the absorbed water allowed resolution of the water into four different states. The amounts of water in these states were different for samples before and after heat denaturation. In the case of denatured beta-lactoglobulin, a smaller amount of water with thermal properties different from ordinary water was observed and its total water binding capacity was lower. The thermal stability of beta-lactoglobulin in the water content range from 0 to 0.75 g/g showed a strong dependence on the degree of hydration. A correlation was observed between the changes in the thermal stability of the protein and the changes in the state of the absorbed water. The results are compared with those obtained from similar measurements of other globular proteins and of fibrillar proteins.     

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.     

Differences in the processes of beta-lactoglobulin cold and heat denaturations.

The changes in beta-lactoglobulin upon cold and heat denaturation were studied by scanning calorimetry, CD, and NMR spectroscopy. It is shown that, in the presence of urea, these processes of beta-lactoglobulin denaturation below and above 308 K are accompanied by different structural and thermodynamic changes. Analysis of the NOE spectra of beta-lactoglobulin shows that changes in the spin diffusion of beta-lactoglobulin after disruption of the unique tertiary structure upon cold denaturation are much more substantial than those upon heat denaturation. In cold denatured beta-lactoglobulin, the network of residual interactions in hydrophobic and hydrophilic regions of the molecules is more extensive than after heat denaturation. This suggests that upon cold- and heat-induced unfolding, the molecule undergoes different structural rearrangements, passing through different denaturation intermediates. From this point of view, cold denaturation can be considered to be a two stage process with a stable intermediate. A similar equilibrium intermediate can be obtained at 35 degrees C in 6.0 M urea solution, where the molecule has no tertiary structure. Cooling or heating of the solution from this temperature leads to unfolding of the intermediate. However, these processes differ in cooperativity, showing noncommensurate sigmoidal-like changes in efficiency of spin diffusion, ellipticity at 222 nm, and partial heat capacity. The disruption with cooling is accompanied by cooperative changes in heat capacity, whereas with heating the heat capacity changes only gradually. Considering the sigmoidal shape of the heat capacity change an extended heat absorption peak, we propose that the intermediate state is stabilized by enthalpic interactions.     

Stable monomeric intermediate with exposed Cys-119 is formed during heat denaturation of beta-lactoglobulin

The role of the free sulfhydryl group of beta-lactoglobulin in the formation of a stable non-native monomer during heat-treatment of beta-lactoglobulin solutions was investigated. Two concomitant events occurred at the earlier stage of heating: unfolding of native globular monomer and intramolecular sulfhydryl/disulfide exchange reaction. Thus, two denatured monomeric species were formed: a non-native monomer with exposed Cys-121 (Mcys121) which became reversible after cooling, and a stable non-native monomer with exposed Cys-119 (Mcys119) which exhibited both a larger hydrodynamic conformation than native monomer and low solubility at pH 4.7. The results also show that the formation of these monomeric species throughout heat-induced denaturation of native beta-lg monomers is faster than their subsequent aggregation. A mechanism describing the behavior of beta-lg denaturation/aggregation during heat-treatment under selected conditions (5.8 mg/ml, low ionic strength, pH 6.6, 85 degrees C) is presented.     

Kinetic and thermodynamic parameters for heat denaturation of human recombinant lactoferrin from rice

Heat denaturation of recombinant human lactoferrin (rhLf) from rice with 3 different iron-saturation degrees, holo rhLf (iron-saturated), AsIs rhLf (60% iron saturation), and apo rhLf (iron-depleted), was studied. The 3 forms of rhLf were subjected to heat treatment, and the kinetic and thermodynamic parameters of the denaturation process were determined. Thermal denaturation of rhLf was assessed by measuring the loss of reactivity against specific antibodies. D(t) values (time to reduce 90% of immunoreactivity) decreased with increasing temperature of treatment for apo and holo rhLf, those values being higher for the iron-saturated form, which indicates that iron confers thermal stability to rhLf. However, AsIs rhLf showed a different behaviour with an increase in resistance to heat between 79 °C and 84 °C, so that the kinetic parameters could not be calculated. The heat denaturation process for apo and holo rhLf was best described assuming a reaction order of 1.5. The activation energy of the denaturation process was 648.20 kJ/mol for holo rhLf and 406.94 kJ/mol for apo rhLf, confirming that iron-depleted rhLf is more sensitive to heat treatment than iron-saturated rhLf.     

Calorimetric study of heat denaturation of toxins

A calorimetric study of reversible heat denaturation of cytotoxin I, neurotoxins I and II in aqueous solution has been carried out. All of them are low molecular proteins from snake venom. Thermodynamic parameters of the transition of the toxins from native to denatured state were determined. Temperature dependences of a specific enthalpy delta dH of the transition were found. It was shown, that upon denaturation the changes in the value of partial heat capacity delta dCp for each of the toxins were constant and did not depend on medium conditions, i.e. composition of a solvent, pH and temperature of the transition. The results of the calorimetric study of the toxins are discussed along with structural peculiarities of low molecular weight proteins (less than 10 000 D) characterized by the amount of van der Waals' interactions between non-polar groups and intramolecular hydrogen bonds.     

Structural stability of beta-lactoglobulin in the presence of kosmotropic salts. A kinetic and thermodynamic study

The thiol group of beta-lactoglobulin reacted very sluggishly with dithio-bis-nitro-benzoic acid as compared to that of glutathione at pH 6.85. The pKapp value of the thiol group of the protein was 9.35. In the presence of 3 M urea, the thiol group reacted completely with dithio-bis-nitrobenzoic acid at pH 6.85. Heating (from 50 degrees to 80 degrees) increased the exposure of the thiol by dissociating the dimer unit. From the pseudo-first order rate constants of heat-exposure of thiol, thermodynamic activation parameters, delta G++, delta H++, and delta S++, for the heat-dissociation of beta-lactoglobulin dimer were estimated to be 23,290 cal/mol, 31,160 cal/mol, and 22.9 e.u. (at 70 degrees), respectively. Addition of kosmotropic salts, chloride, tartrate, sulfate, phosphate, and citrate (0.2 M) decreased the heat-induced exposure of the thiol group (at 70 degrees), probably by decreasing the dissociation of the dimer at pH 6.85. The relative change in free energy of activation for the dissociation of the dimer, delta(delta G++dimer), in the presence of the salts was positive, suggesting that these additives increase the stability of the dimer against heat. These salts also increased the conformational stability of beta-lactoglobulin as revealed by an increase in -delta(delta G0conf) values in their presence. Both delta(delta G++dimer) and -delta(delta G0conf) values followed the order, chloride less than tartrate less than sulfate less than phosphate less than citrate. These salts seem to manifest their structure-stabilizing effect by increasing both inter- and intramolecular hydrophobic interactions via changes in structure of water.     

Protein heat capacity: inconsistencies in the current view of cold denaturation

The present study shows on the basis of the thermodynamic stability criterion (partial differential S/partial differential T)p>0 that partitioning of the entropy of cold-unfolding of a protein into independent positive conformational and negative hydrational contributions is incorrect. Furthermore it provides a new microscopic interpretation of protein heat capacity that takes into account the significant fluctuations in energy and entropy which result from the small size of these macromolecules.     

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.     

Quantitative and thermodynamic study of weak A erythrocyte phenotypes]

The analysis of more than 140 "weak A" samples: A3, Ax, Aend, Am, Ay and Ael, support the classical distinction between each subgroup which has been established on serological and genetical data. Accordingly, a valuable classification of these rare phenotypes must take into account, (i) the mode of inheritance, (ii) the agglutination pattern of the RBC by anti-A reagents, (iii) the presence or absence of soluble A substances in the saliva of secretors. The question is then open to know if such related erythrocytic antigens, whose specificity appears to be very similar, could be described on a quantitative basis or on qualitative structural variations. Evidence for quantitative differences was first demonstrated by a gradual decrease in the standard agglutinability of "weak A" RBC with human anti-A (B) sera, from A3 red cells (63 +/- 10%) to Ax (33 +/- 10%), Aend (10 +/- 5%) then Am, Ay and Ael (0%), and secondly by direct measurement of A antigen site densities, the mean values being respectively 35.10(3) A sites/RBC (A3); 4.8 10(3) (Ax); 3.5 10(3) (Aend) and 0.7 10(3) (Am, Ael). Further investigation on A3, Ax and Aend RBC agglutinability lead also to the demonstration of a large heterogeneity in the A antigenic content of red cells inside one individual sample. The most striking result was obtained with Aend phenotypes which appeared like A + O transmitted mosaicisms. However, heterogeneity was also observed, but to a lesser extent, among A3 and Ax RBC. The significance of this heterogeneity is discussed and used to explained the typical picture of agglutinability commonly observed with such red cells and anti-A antibodies. Qualitative difference were also studied by estimation of equilibrium constants (Ko) and thermodynamic parameters (delta Fo, delta Ho and delta So) associated with the binding of rabbit 125I-IgG anti-A molecules onto A RBC determinants. Only small variations of thermodynamic parameters were observed between each subgroup, but the high Ko values (greater than 10(8)M-1) measured, strongly suggest that "weak A" RBC determinants would process a common antigenic structure of the type: alpha-GalNAc (1 leads to 3) [alphaLFuc (1 leads to 2) beta Gal. However, the small differences of reactivity observed from one sample to an other could be related to slight variations in tridimensional configurations of oligosaccharides chains bearing the A specificity, associated with their variable antigenic content.     

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.     

The heat denaturation of bacterial ribonuclease studied by infrared spectroscopy]

The thermal denaturation of bacterial ribonuclease in the interval of pH 2.5-7.0 has been investigated by means of infra-red spectroscopy method. The protein melting for pH 2.5 begins at the temperature 25 degrees C and is accompanied by secondary protein structure reconstruction, partially destroying native beta-structure and leading to new denatured conformation appearance of different types of beta-turns. Spectral changes for pH 3.5 and 7.0 are significantly less in the same frequency areas. At the temperature more than 50 degrees C protein aggregation takes place with inter-molecule-beta-form formation.     

Linear free-energy model description of the conformational stability of uracil-DNA glycosylase inhibitor A thermodynamic characterization of interaction with denaturant and cold denaturation.

The equilibrium unfolding of uracil DNA glycosylase inhibitor (Ugi), a small acidic protein of molecular mass 9474 Da, has been studied by a combination of thermal-induced and guanidine hydrochloride (GdnCl)-induced denaturation. The analysis of the denaturation data provides a measure of the changes in conformational free energy, enthalpy, entropy and heat capacity DeltaCp that accompany the equilibrium unfolding of Ugi over a wide range of temperature and GdnCl concentration. The unfolding of Ugi is a simple two-state, reversible process. The protein undergoes both low-temperature and high-temperature unfolding even in the absence of GdnCl but more so in the presence of denaturant. The data are consistent with the linear free-energy model and with a temperature independent DeltaCp over the large temperature range of unfolding. The small DeltaCp (6.52 kJ.mol-1.K-1) for the unfolding of Ugi, is perhaps a reflection of a relatively small, buried hydrophobic core in the folded form of this small monomeric protein. Despite a relatively low value of DeltaG(H2O), 7.40 kJ.mol-1 at pH 8.3, Ugi displays considerable stability with the temperature of maximum stability being 301.6 K.     

Lymphocyte chromatin changes in Down's disease detected by heat denaturation]

By using fluorescent microscopy and acridine orange staining it was shown in the studies on short-term culture of human cells that the melting patterns of chromatin DNA of intact lymphocytes of healthy individuals represented the curves with 6 maxima (F530) at the temperature ranges of 45 degrees C, 65 degrees C, 85 degrees C, and 92 degrees C (P less than 0.01). The melting patterns of lymphocytes from patients with Down's disease represented curves with 4 maxima at the temperature ranges of 65 degrees C, 85 degrees C, 88 degrees C, 92 degrees C (P less than 0.01). No decline in the fluorescence intensity at the temperature intervals of 78 degrees C-85 degrees C was apparently due to a greater degree of condensation of definite regions of the trisomal cell chromatin complex. Possible mechanisms accounting for structural readjustments of the interphasic human lymphocyte chromatin occurring under thermal effects are discussed.     

Enhanced thermodynamic stability of beta-lactoglobulin at low pH. A possible mechanism.

The thermodynamic stability of beta-lactoglobulin (beta-Lg) was studied at acidic and near-neutral pH values using equilibrium thermal-unfolding measurements. Transition temperature increased with a decrease in pH from 7.5 to 6.5 and 3.0 to 1.5, suggesting an increase in the net protein stability. Determination of the change in free energy of unfolding and extrapolation into the nontransition region revealed that beta-Lg increases its stability by increasing the magnitude of the change in free energy of unfolding at the temperature of maximum stability, as well as by increasing the temperature of maximum stability. The relative difference in the change in free energy of unfolding at 70 degrees C (with a reference pH of 7.5) was positive and its magnitude increased with a decrease in pH from 7.0 to 1.5 van't Hoff plots of thermal unfolding of beta-Lg at all pH values studied were non-linear and the measured changes in the enthalpy and entropy of unfolding for beta-Lg were high and positive. The relative magnitude of change of both enthalpy and entropy at 70 degrees C (compared with pH 7.5) increased with a decrease in pH up to 1.5. A possible mechanism for the increased stability of beta-Lg at low pH is discussed.