The peculiar nature of the guanidine hydrochloride-induced two-state denaturation of staphylococcal nuclease: a calorimetric study.

This work determines the ratio of DeltaH(vH) /DeltaH(cal) for staphylococcal nuclease (SN) denaturation in guanidine hydrochloride (GdnHCl) to test whether GdnHCl-induced denaturation is two-state. Heats of mixing of SN as a function of [GdnHCl] were determined at pH 7.0 and 25 degrees C. The resulting plot of DeltaH(mix) vs [GdnHCl] exhibits a sigmoid shaped curve with linear pre- and post-denaturational base lines. Extending the pre- and post-denaturational lines to zero [GdnHCl] gives a calorimetric DeltaH (DeltaH(cal)) of 24.1 +/- 1.0 kcal/mol, for SN denaturation in the limit of zero GdnHCl concentration. Guanidine hydrochloride-induced denaturation Gibbs energy changes in the limit of zero denaturant concentration (DeltaG degrees (N)(-)(D)) at pH 7. 0 were determined for SN from fluorescence measurements at fixed temperatures over the range from 15 to 35 degrees C. Analysis of the resulting temperature-dependent DeltaG degrees (N)(-)(D) data defines a van't Hoff denaturation enthalpy change (DeltaH(vH)) of 26. 4 +/- 2.8 kcal/mol. The model-dependent van't Hoff DeltaH(vH) divided by the model-independent DeltaH(cal) gives a ratio of 1.1 +/- 0.1 for DeltaH(vH)/DeltaH(cal), a result that rules out the presence of thermodynamically important intermediate states in the GdnHCl-induced denaturation of SN. The likelihood that GdnHCl-induced SN denaturation involves a special type of two-state denaturation, known as a variable two-state process, is discussed in terms of the thermodynamic implications of the process.     

Compatibility of osmolytes with Gibbs energy of stabilization of proteins

This study led to the conclusion that naturally occurring osmolytes which are known to protect proteins against denaturing stresses, do not perturb the Gibbs energy of stabilization of proteins at 25 degrees C (DeltaG(D) degrees ) which has been shown to control the in vivo rate of degradative protein turnover (Pace et al., Acta Biol. Med. Germ 40 (1981) 1385-1392). This conclusion has been reached from our studies of heat-induced denaturation of lysozyme, ribonuclease A, cytochrome c and myoglobin in the presence of different concentrations of osmolytes, namely, glycine, proline, sarcosine and glycine-betaine. At a fixed concentration of osmolyte a heat-induced denaturation curve measured by following changes in the molar absorption coefficient of the protein, was analyzed for T(m), the midpoint of the denaturation and DeltaH(m), the enthalpy change of denaturation at T(m). Values of DeltaG(D) degrees were determined with Gibbs-Helmoltz equation using known values of T(m), DeltaH(m) and DeltaC(p), the constant-pressure heat capacity change. It has been observed that T(m) increases with the osmolyte concentration, whereas DeltaG(D) degrees remains unaffected in the presence of the osmolyte. This observation on DeltaG(D) degrees in the presence of osmolytes has been considered in the physiological context.     

Effect of monomeric and oligomeric sugar osmolytes on ΔGD, the Gibbs energy of stabilization of the protein at different pH values: Is the sum effect of monosaccharide individually additive in a mixture?

Thermal denaturation curves of ribonuclease-A were measured by monitoring changes in the far-UV circular dichroism (CD) spectra in the presence of different concentrations of six sugars (glucose, fructose, galactose, sucrose, raffinose and stachyose) and mixture of monosaccharide constituents of each oligosaccharide at various pH values in the range of 6.0-2.0. These measurements gave values of T(m) (midpoint of denaturation), DeltaH(m) (enthalpy change at T(m)), DeltaC(p) (constant-pressure heat capacity change) under a given solvent condition. Using these values of DeltaH(m), T(m) and DeltaC(p) in appropriate thermodynamic relations, thermodynamic parameters at 25 degrees C, namely, DeltaG(D)(o) (Gibbs energy change), DeltaH(D)(o) (enthalpy change), and DeltaS(D)(o) (entropy change) were determined at a given pH and concentration of each sugar (including its mixture of monosaccharide constituents). Our main conclusions are: (i) each sugar stabilizes the protein in terms of T(m) and DeltaG(D)(o), and this stabilization is under enthalpic control, (ii) the protein stabilization by the oligosaccharide is significantly less than that by the equimolar concentration of the constituent monosaccharides, and (iii) the stabilization by monosaccharides in a mixture is fully additive. Furthermore, measurements of the far- and near-UV CD spectra suggested that secondary and tertiary structures of protein in their native and denatured states are not perturbed on the addition of sugars.     

Thermal stability of acetylcholinesterase from Bungarus fasciatus venom as investigated by capillary electrophoresis

Previous studies on the conformation of the monomeric acetylcholinesterase (AChE) from the krait (Bungarus fasciatus) venom showed that the protein possesses a large permanent dipole moment. These studies predicted that thermal irreversible denaturation must occur via partially unfolded states. The thermal stability of Bungarus AChE was determined using capillary electrophoresis (CE) with optimized conditions. Runs performed at convenient temperature scanning rates provided evidence for an irreversible denaturation process according to the Lumry and Eyring model. The mid-transition temperature, T(m), and the effective enthalpy change, DeltaH(m) were determined at different pH. The temperature dependence of the free energy, DeltaG, of Bungarus AChE unfolding was drawn using values of T(m), DeltaH(m) and DeltaC(p) determined by CE. The thermodynamic parameters for the thermal denaturation of the monomeric snake enzyme were compared with those of different dimeric and tetrameric ChEs. It was shown that the changes in the ratio of DeltaH(cal/)DeltaH(vH) and DeltaC(p) reflect the oligomerization state of these proteins. All these results indicate that wild-type monomeric Bungarus AChE is a stable enzyme under standard conditions. However, designed mutants of this enzyme capable of degrading organophosphates have to be engineered to enhance their thermostability.     

Thermostabilization of ovotransferrin by anions for pasteurization of liquid egg white

The effects of anions on the thermostability of ovotransferrin (oTf) were investigated. The temperature, T(m), causing aggregation of oTf was measured in the presence or absence of anions, and the denaturation temperature, T(m)(DSC), was also determined by differential scanning calorimetry (DSC) in the presence of the citrate anion. We found that some anions (phosphate, sulfate and citrate) raised temperature T(m) of oTf by about 5-7 degrees C. However, neither sodium chloride nor sodium bicarbonate raised T(m) by that much. Temperature T(m) was increased by increasing the concentration of the citrate anion, and was in good agreement with denaturation temperature T(m)(DSC), suggesting that denaturation of the oTf molecules resulted in aggregation of oTf. We also demonstrated that the anions, especially sulfate, repressed the heat-aggregation of liquid egg white.The Van't Hoff plot from the T(m) and DeltaH(d) values revealed that two anion-binding sites were concerned with heat stabilization. These binding sites may have been concerned with sulfate binding (not bicarbonate binding) that is found in the crystal structure of apo-form of oTf, since the bicarbonate anion did not raise T(m).     

Possibility for discriminating between two representative non two-state thermal unfolding models of proteins by DSC

Possible differences between two representative non two-state thermal unfolding mechanisms of protein are discussed concerning differential scanning calorimetry. Numerical simulations showed that, by DSC measurement, it is hard to discriminate between the independent model, which assumes independent unfolding domains in a protein, and the sequential model, which assumes intermediate(s) between native and denatured states, especially when values of molecular weight, denaturation enthalpy, and difference in denaturation temperature of each denaturation process are large. DSC curve analysis of Aspergillus niger glucoamylase based on these two models gave essentially the same thermodynamic parameters.     

Thermodynamic characterization of the osmolyte- and ligand-folded states of Bacillus subtilis ribonuclease P protein

In Bacillus subtilis, P protein is the noncatalytic component of ribonuclease P (RNase P) that is critical for achieving maximal nuclease activity under physiological conditions. P protein is predominantly unfolded (D) at neutral pH and low ionic strength; however, it folds upon the addition of sulfate anions (ligands) as well as the osmolyte trimethylamine N-oxide (TMAO) [Henkels, C. H., Kurz, J. C., Fierke, C. A., and Oas, T. G. (2001) Biochemistry 40, 2777-2789]. Since the molecular mechanisms that drive protein folding for these two solutes are different, CD thermal denaturation studies were employed to dissect the thermodynamics of protein unfolding from the two folded states. A global fit of the free-energy of TMAO-folded P protein versus [TMAO] and temperature yields T(S), DeltaH(S), and DeltaC(p) of unfolding for the poorly populated, unliganded, folded state (N) in the absence of TMAO. These thermodynamic parameters were used in the fit of the data from the coupled unfolding/ligand dissociation reaction to obtain the sulfate dissociation constant (K(d)) and the DeltaH and DeltaC(p) of dissociation. These fits yielded a DeltaC(p) of protein unfolding of 826 +/- 23 cal mol(-)(1) K(-)(1) and a DeltaC(p) of 1554 +/- 29 cal mol(-)(1) K(-)(1) for the coupled unfolding and dissociation reaction (NL(2) --> D + 2L). The apparent stoichiometry of sulfate binding is two, so the DeltaC(p) increment of ligand dissociation is 363 +/- 9 cal mol(-)(1) K(-)(1) per site. Because N and NL(2) appear to be structurally similar and therefore similarly solvated using standard biophysical analyses, we attribute a substantial portion of this DeltaC(p) increment to an increase in conformational heterogeneity coincident with the NL(2) --> N + 2L transition.     

Differential scanning calorimetric studies on binding of N-acetyl-D-glucosamine to lysozyme

Differential scanning calorimetric (DSC) measurements were performed on the thermal denaturation of lysozyme and lysozyme complexed with N-acetyl-D-glucosamine (GlcNAc) at pH 5.00 (acetate buffer), 4.25 and 2.25 (Gly-HCl buffer). DSC data have been analyzed to obtain denaturation temperature T(d), enthalpy of denaturation DeltaH(D), heat capacity of denaturation DeltaC(pd) and cooperativity index eta. From these thermodynamic parameters, the binding constant K(L) and enthalpy of binding DeltaH(L), for the weak binding of lysozyme with GlcNAc have been determined. The values of K(L) and DeltaH(L) at pH 5.00 and 298 K are 42 +/- 4 M(-1) and -24 +/- 4 kJ mol(-1), respectively, and agree very well with the experimentally determined values from equilibrium and other studies. The binding constant has also been estimated by simulating the DSC curve with varying values of K(L) (T(d)) until it matches the experimental curve.     

Acid-induced changes in thermal stability and fusion activity of influenza hemagglutinin

The conformational and thermal stability of full-length hemagglutinin (HA) of influenza virus (strain X31) has been investigated using a combination of differential scanning calorimetry (DSC), analytical ultracentrifugation, fluorescence, and circular dichroism (CD) spectroscopy as a function of pH. HA sediments as a rosette comprised of 5-6 trimers (31-35 S) over the pH range of 7.4-5.4. The DSC profile of HA in the native state at pH 7.4 is characterized by a single cooperative endotherm with a transition temperature (Tm) of 66 degrees C and unfolding enthalpy (DeltaH(cal)) of 800 kcal x (mol of trimer)(-1). Upon acidification to pH 5.4, there is a significant decrease in the transition temperature (from 66 to 45 degrees C), unfolding enthalpy [from 800 to 260 kcal x (mol of trimer)(-1)], and DeltaH(cal)/DeltaH(vH) ratio (from 3.0 to approximately 1.3). Whereas the far- and near-UV ellipticities are maintained over this pH range, there is an acid-induced increase in surface hydrophobicity and decrease in intrinsic tryptophanyl fluorescence. The major contribution to the DSC endotherm arises from unfolding HA1 domains. The relationship between acid-induced changes in thermal stability and the fusion activity of HA has been examined by evaluating the kinetics and extent of fusion of influenza virus with erythrocytes over the temperature and pH range of the DSC measurements. Surprisingly, X31 influenza virus retains its fusion activity at acidic pH and temperatures significantly below the unfolding transition of HA. This finding is consistent with the notion that the fusion activity of influenza virus may involve structural changes of only a small fraction of HA molecules.     

A new method for determining the constant-pressure heat capacity change associated with the protein denaturation induced by guanidinium chloride (or urea)

Differential scanning calorimetry (DSC) provides authentic and accurate value of DeltaC(p)(X), the constant-pressure heat capacity change associated with the N (native state)<-->X (heat denatured state), the heat-induced denaturation equilibrium of the protein in the absence of a chemical denaturant. If X retains native-like buried hydrophobic interaction, DeltaC(p)(X) must be less than DeltaC(p)(D), the constant-pressure heat capacity change associated with the transition, N<-->D, where the state D is not only more unfolded than X but it also has its all groups exposed to water. One problem is that for most proteins D is observed only in the presence of chemical denaturants such as guanidinium chloride (GdmCl) and urea. Another problem is that DSC cannot yield authentic DeltaC(p)(D), for its measurement invokes the existence of putative specific binding sites for the chemical denaturants on N and D. We have developed a non-calorimetric method for the measurements of DeltaC(p)(D), which uses thermodynamic data obtained from the isothermal GdmCl (or urea)-induced denaturation and heat-induced denaturation in the presence of the chemical denaturant concentration at which significant concentrations of both N and D exist. We show that for each of the proteins (ribonuclease-A, lysozyme, alpha-lactalbumin and chymotrypsinogen) DeltaC(p)(D) is significantly higher than DeltaC(p)(X). DeltaC(p)(D) of the protein is also compared with that estimated using the known heat capacities of amino acid residues and their fractional area exposed on denaturation.     

Thermodynamic stability of ribonuclease A in alkylurea solutions and preferential solvation changes accompanying its thermal denaturation: a calorimetric and spectroscopic study

The effect of methylurea, N,N'-dimethylurea, ethylurea, and butylurea as well as guanidine hydrochloride (GuHCl), urea and pH on the thermal stability, structural properties, and preferential solvation changes accompanying the thermal unfolding of ribonuclease A (RNase A) has been investigated by differential scanning calorimetry (DSC), UV, and circular dichroism (CD) spectroscopy. The results show that the thermal stability of RNase A decreases with increasing concentration of denaturants and the size of the hydrophobic group substituted on the urea molecule. From CD measurements in the near- and far-UV range, it has been observed that the tertiary structure of RNase A melts at about 3 degrees C lower temperature than its secondary structure, which means that the hierarchy in structural building blocks exists for RNase A even at conditions at which according to DSC and UV measurements the RNase A unfolding can be interpreted in terms of a two-state approximation. The far-UV CD spectra also show that the final denatured states of RNase A at high temperatures in the presence of different denaturants including 4.5 M GuHCl are similar to each other but different from the one obtained in 4.5 M GuHCl at 25 degrees C. The concentration dependence of the preferential solvation change delta r23, expressed as the number of cosolvent molecules entering or leaving the solvation shell of the protein upon denaturation and calculated from DSC data, shows the same relative denaturation efficiency of alkylureas as other methods.     

Differential scanning calorimetric studies of a Bacillus halodurans alpha-amylase

The thermal unfolding of Amy 34, a recombinant alpha-amylase from Bacillus halodurans, has been investigated using differential scanning calorimetry (DSC). The denaturation of Amy 34 involves irreversible processes with an apparent denaturation temperature (T(m)) of 70.8 degrees C at pH 9.0, with four transitions, as determined using multiple Gaussian curves. The T(m) increased by 5 degrees C in the presence of 100-fold molar excess of CaCl2 while the aggregation of Amy 34 was observed in the presence of 1000-fold molar excess of CaCl2. Increase in the calcium ion concentration from 1- to 5-fold molar excess resulted in an increase in calorimetric enthalpy (DeltaH(cal)), however, at higher concentrations of CaCl2 (up to 100-fold), DeltaH(cal) was found to decrease, accompanied by a decrease in entropy change (DeltaS), while the T(m) steadily increased. The presence of 100-fold excess of metal chelator, EDTA, resulted in a decrease in T(m) by 10.4 degrees C. T(m) was also decreased to 61.1 degrees C and 65.9 degrees C at pH 6.0 and pH 11.0, respectively.     

Enzyme thermoinactivation in anhydrous organic solvents

Three unrelated enzymes (ribonuclease, chymotrypsin, and lysozyme) display markedly enhanced thermostability in anhydrous organic solvents compared to that in aqueous solution. At 110-145 degrees C in nonaqueous media all three enzymes inactivate due to heat-induced protein aggregation, as determined by gel filtration chromatography. Using bovine pancreatic ribonuclease A as a model, it has been established that enzymes are much more thermostable in hydrophobic solvents (shown to be essentially inert with respect to their interaction with the protein) than in hydrophilic ones (shown to strip water from the enzyme). The heat-induced aggregates of ribonuclease were characterized as both physically associated and chemically crosslinked protein agglomerates, with the latter being in part due to transamidation and intermolecular disulfide interchange reactions. The thermal denaturation of ribonuclease in neat organic solvents has been examined by means of differential scanning calorimetry. In hydrophobic solvents, the enzyme exhibits greatly enhanced thermal denaturation temperatures (T(m) values as high as 124 degrees C) compared to aqueous solution. The thermostability of ribonuclease towards heat-induced denaturation and aggregation decreases as the water content of the protein powder increases. The experimental data obtained suggest that enzymes are extremely thermostable in anhydrous organic solvents due to their conformational rigidity in the dehydrated state and their resistance to nearly all the covalent reactions causing irreversible thermoinactivation of enzymes in aqueous solution.     

Difference in the mechanisms of the cold and heat induced unfolding of thioredoxin h from Chlamydomonas reinhardtii: spectroscopic and calorimetric studies

The thermodynamic stability and temperature induced structural changes of oxidized thioredoxin h from Chlamydomonas reinhardtii have been studied using differential scanning calorimetry (DSC), near- and far-UV circular dichroism (CD), and fluorescence spectroscopies. At neutral pH, the heat induced unfolding of thioredoxin h is irreversible. The irreversibly unfolded protein is unable to refold due to the formation of soluble high-order oligomers. In contrast, at acidic pH the heat induced unfolding of thioredoxin h is fully reversible and thus allows the thermodynamic stability of this protein to be characterized. Analysis of the heat induced unfolding at acidic pH using calorimetric and spectroscopic methods shows that the heat induced denaturation of thioredoxin h can be well approximated by a two-state transition. The unfolding of thioredoxin h is accompanied by a large heat capacity change [6.0 +/- 1.0 kJ/(mol.K)], suggesting that at low pH a cold denaturation should be observed at the above-freezing temperatures for this protein. All used methods (DSC, near-UV CD, far-UV CD, Trp fluorescence) do indeed show that thioredoxin h undergoes cold denaturation at pH <2.5. The cold denaturation of thioredoxin h cannot, however, be fitted to a two-state model of unfolding. Furthermore, according to the far-UV CD, thioredoxin h is fully unfolded at pH 2.0 and 0 degrees C, whereas the other three methods (near-UV CD, fluorescence, and DSC) indicate that under these conditions 20-30% of the protein molecules are still in the native state. Several alternative mechanisms explaining these results such as structural differences in the heat and cold denatured state ensembles and the two-domain structure of thioredoxin h are discussed.     

The stability, structural organization, and denaturation of pectate lyase C, a parallel beta-helix protein

Pectate lyase C (pelC) was the first protein in which the parallel beta-helix structure was recognized. The unique features of parallel beta-helix-containing proteins-a relatively simple topology and unusual interactions among side chains-make pelC an interesting protein to study with respect to protein folding. In this paper, we report studies of the unfolding equilibrium of pelC. PelC is unfolded reversibly by gdn-HCl at pH 7 and 5, as monitored by far- and near-UV CD and fluorescence. The coincidence of these spectroscopically detected transitions is consistent with a two-state transition at pH 7, but the three probes are not coincident at pH 5. No evidence was found for a loosely folded intermediate in the transition region at pH 5. At pH 7, the for unfolding is 12.2 kcal/mol, with the midpoint of the transition at 0.99 M gdn-HCl and m = 12.3 kcal/(mol.M). Thus, pelC is unusually stable and has an m value that is much larger than for typical globular proteins. Thermal denaturation of pelC has been studied by differential scanning calorimetry (DSC) and by CD. Although thermal denaturation is not reversible, valid thermodynamic data can be obtained for the unfolding transition. DeltaH(van't Hoff)/DeltaH(cal) is less than 1 for pHs between 5 and 8, with a maximum value of 0.91 at pH 7 decreasing to 0.85 at pH 8 and to 0.68 at pH 5. At all pHs studied, the excess heat capacity can be deconvoluted into two components corresponding to two-state transitions that are nearly coincident at pH 7, but deviate more at higher and lower pH. Thus, pelC appears to consist of two domains that interact strongly and unfold in a cooperative fashion at pH 7, but the cooperativity decreases at higher and lower pH. The crystal structure of pelC shows no obvious domain structure, however.     

Using a second‐order differential model to fit data without baselines in protein isothermal chemical denaturation

In vitro protein stability studies are commonly conducted via thermal or chemical denaturation/renaturation of protein. Conventional data analyses on the protein unfolding/(re)folding require well‐defined pre‐ and post‐transition baselines to evaluate Gibbs free‐energy change associated with the protein unfolding/(re)folding. This evaluation becomes problematic when there is insufficient data for determining the pre‐ or post‐transition baselines. In this study, fitting on such partial data obtained in protein chemical denaturation is established by introducing second‐order differential (SOD) analysis to overcome the limitations that the conventional fitting method has. By reducing numbers of the baseline‐related fitting parameters, the SOD analysis can successfully fit incomplete chemical denaturation data sets with high agreement to the conventional evaluation on the equivalent completed data, where the conventional fitting fails in analyzing them. This SOD fitting for the abbreviated isothermal chemical denaturation further fulfills data analysis methods on the insufficient data sets conducted in the two prevalent protein stability studies.     

Kinetically robust monomeric protein from a hyperthermophile

Equilibrium and kinetic studies were carried out under denaturation conditions to clarify the energetic features of the high stability of a monomeric protein, ribonuclease HII, from a hyperthermophile, Thermococcus kodakaraensis (Tk-RNase HII). Guanidine hydrochloride (GdnHCl)-induced unfolding and refolding were measured with circular dichroism at 220 nm, and heat-induced denaturation was studied with differential scanning calorimetry. Both GdnHCl- and heat-induced denaturation are very reversible. It was difficult to obtain the equilibrated unfolding curve of Tk-RNase HII below 40 degrees C, because of the remarkably slow unfolding. The two-state unfolding and refolding reactions attained equilibrium at 50 degrees C after 2 weeks. The Gibbs energy change of GdnHCl-induced unfolding (DeltaG(H(2)O)) at 50 degrees C was 43.6 kJ mol(-1). The denaturation temperature in the DSC measurement shifted as a function of the scan rate; the denaturation temperature at a scan rate of 90 degrees C h(-1) was higher than at a scan rate of 5 degrees C h(-1). The unfolding and refolding kinetics of Tk-RNase HII were approximated as a first-order reaction. The ln k(u) and ln k(r) values depended linearly on the denaturant concentration between 10 and 50 degrees C. The DeltaG(H(2)O) value obtained from the rate constant in water using the two-state model at 50 degrees C, 44.5 kJ mol(-1), was coincident with that from the equilibrium study, 43.6 kJ mol(-1), suggesting the two-state folding of Tk-RNase HII. The values for the rate constant in water of the unfolding for Tk-RNase HII were much smaller than those of E. coli RNase HI and Thermus thermophilus RNase HI, which has a denaturation temperature similar to that of Tk-RNase HII. In contrast, little difference was observed in the refolding rates among these proteins. These results indicate that the stabilization mechanism of monomeric protein from a hyperthermophile, Tk-RNase HII, with reversible two-state folding is characterized by remarkably slow unfolding.     

Thermal unfolding of soybean peroxidase. Appropriate high denaturant concentrations induce cooperativity allowing the correct measurement of thermodynamic parameters

We have earlier reported that both guanidine hydrochloride (GdnHCl)-induced and heat-induced unfolding of seed coat soybean peroxidase (SBP), monitored by far UV CD, show single step transition. However, although GdnHCl-induced unfolding follows a two-state pathway, the heat-induced denaturation proceeds through intermediates as indicated by the very low cooperativity of transition. In the former case, analysis of the data based on the two-state model gives true thermodynamic parameters, whereas underestimated values are obtained in the latter case. Available complex equations also cannot be applied for the analysis of the thermal unfolding of SBP due to the absence of separate transitions for the intermediates. In the present study, we report a method to obtain true thermodynamic parameters from thermal transition curves of SBP using the two-state model. When SBP is subjected to thermal unfolding at high GdnHCl concentrations (5.8-6.9 M), cooperative behavior is observed, which allowed the analysis by the two-state model to determine their thermodynamic parameters. We then obtained the thermodynamic parameters in the absence of GdnHCl by extrapolating the graph of linear dependence of DeltaH(m) on T(m) to the T(m) corresponding to 0 m GdnHCl. Another key point for checking the validity of our method was the fact that the unfolded state of SBP generated by either heat or GdnHCl is the same by which we could cross-check our results with that obtained from GdnHCl unfolding. Having obtained the true thermodynamic parameters, we report a detailed thermodynamic study of SBP. Further we address the effect of heme in the thermal unfolding mechanism of SBP.     

Physical-chemical characterization and stability study of alpha-trypsin at pH 3.0 by differential scanning calorimetry

alpha-Trypsin is a serine-protease with a polypeptide chain of 223 amino acid residues and six disulfide bridges. It is a globular protein with predominance of antiparallel ss-sheet secondary structure and it has two domains with similar structures. In the present work, a stability study of alpha-trypsin in the acid pH range was performed and some physical-chemical denaturation parameters were measured by using differential scanning calorimetry (DSC). The alpha-trypsin has a shelf-life (t(95%)) of about 10 months at pH 3.0 and 4 degrees C and its hydrolysis into the psi-trypsin isoform is negligible during 6 months. The observed ratio DeltaH(cal)/DeltaH(vH) is close to unity, which suggests the occurrence of a two-state transition. At pH 3.0, alpha-trypsin unfolded with T(m) = 325.9 K and DeltaH = 99.10 kcal mol(-1), and the change in heat capacity between the native and unfolded forms of the protein was estimated to be 1.96+/-0.18 kcal mol(-1)K(-1). The stability of alpha-trypsin calculated at 298 K was DeltaG(U)=6.10 kcal mol(-1) at pH 3.0. These values are in the range expected for a small globular protein. These results show that the thermodynamic parameters of unfolding of beta-trypsin do not change substantially after its conversion to alpha-trypsin.