Thermodynamics of unfolding for turkey ovomucoid third domain: thermal and chemical denaturation.

We have used thermal and chemical denaturation to characterize the thermodynamics of unfolding for turkey ovomucoid third domain (OMTKY3). Thermal denaturation was monitored spectroscopically at a number of wave-lengths and data were subjected to van't Hoff analysis; at pH 2.0, the midpoint of denaturation (Tm) occurs at 58.6 +/- 0.4 degrees C and the enthalpy of unfolding at this temperature (delta Hm) is 40.8 +/- 0.3 kcal/mol. When Tm was perturbed by varying pH and denaturant concentration, the resulting plots of delta Hm versus Tm yield a mean value of 590 +/- 120 cal/(mol.K) for the change in heat capacity upon unfolding (delta Cp). A global fit of the same data to an equation that includes the temperature dependence for the enthalpy of unfolding yielded a value of 640 +/- 110 cal/(mol.K). We also performed a variation of the linear extrapolation method described by Pace and Laurents, which is an independent method for determining delta Cp (Pace, C.N. & Laurents, D., 1989, Biochemistry 28, 2520-2525). First, OMTKY3 was thermally denatured in the presence of a variety of denaturant concentrations. Linear extrapolations were then made from isothermal slices through the transition region of the denaturation curves. When extrapolated free energies of unfolding (delta Gu) were plotted versus temperature, the resulting curve appeared linear; therefore, delta Cp could not be determined. However, the data for delta Gu versus denaturant concentration are linear over an extraordinarily wide range of concentrations. Moreover, extrapolated values of delta Gu in urea are identical to values measured directly.     

Urea denaturation of barnase: pH dependence and characterization of the unfolded state.

To investigate the pH dependence of the conformational stability of barnase, urea denaturation curves were determined over the pH range 2-10. The maximum conformational stability of barnase is 9 kcal mol-1 and occurs between pH 5 and 6. The dependence of delta G on urea concentration increases from 1850 cal mol-1 M-1 at high pH to about 3000 cal mol-1 M-1 near pH 3. This suggests that the unfolded conformations of barnase become more accessible to urea as the net charge on the molecule increases. Previous studies suggested that in 8 M urea barnase unfolds more completely than ribonuclease T1, even with the disulfide bonds broken [Pace, C.N., Laurents, D. V., & Thomson, J.A. (1990) Biochemistry 29, 2564-2572]. In support of this, solvent perturbation difference spectroscopy showed that in 8 M urea the Trp and Tyr residues in barnase are more accessible to perturbation by dimethyl sulfoxide than in ribonuclease T1 with the disulfide bonds broken.     

Use of protein unfolding studies to determine the conformational and dimeric stabilities of HIV-1 and SIV proteases.

The free energies of dimer dissociation of the retroviral proteases (PRs) of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) were determined by measuring the effects of denaturants on the protein fluorescence upon the unfolding of the enzymes. HIV-1 PR was more stable to denaturation by chaotropes and extremes of pH and temperature than SIV PR, indicating that the former enzyme has greater conformational stability. The urea unfolding curves of both proteases were sigmoidal and single phase. The midpoints of the transition curves increased with increasing protein concentrations. These data were best described by and fitted to a two-state model in which folded dimers were in equilibrium with unfolded monomers. This denaturation model conforms to cases in which protein unfolding and dimer dissociation are concomitant processes in which folded monomers do not exist [Bowie, J. U., & Sauer, R. T. (1989) Biochemistry 28, 7140-7143]. Accordingly, the free energies of unfolding reflect the stabilities of the protease dimers, which for HIV-1 PR and SIV PR were, respectively, delta GuH2O = 14 +/- 1 kcal/mol (Ku = 39 pM) and 13 +/- 1 kcal/mol (Ku = 180 pM). The binding of a tight-binding, competitive inhibitor greatly stabilized HIV-1 PR toward urea-induced unfolding (delta GuH2O = 19.3 +/- 0.7 kcal/mol, Ku = 7.0 fM). There were also profound effects caused by adverse pH on the protein conformation for both HIV-1 PR and SIV PR, resulting in unfolding at pH values above and below the respective optimal ranges of 4.0-8.0 and 4.0-7.0     

Thermodynamics of the denaturation of pepsinogen by urea.

The denaturation of swine pepsinogen has been studied as a function of urea concentration, pH, and temperature. The unfolding of the protein by urea has been found to be fully reversible under different conditions of pH, temperature, and denaturant concentration. Kinetic experiments have shown that the transition shows two-state behavior at 25 degrees C in the pH range 6-8 covered in this study. Analysis of the equilibrium data obtained at 25 degrees C according to Tanford (Tanford, C. (1970), Adv. Protein Chem. 24, 1) and Pace (Pace, N.C. (1975), Crit. Rev. Biochem. 3, 1) leads to the conclusion that the free energy of stabilization of native pepsinogen, relative to the denatured state, under physiological conditions, is only 6-12 kcal mol-1. The temperature dependence of the equilibrium constant for the unfolding of pepsinogen by urea in the range 20-50 degrees C at pH 8.0 can be described by assigning the following values of thermodynamic parameters for the denaturation at 25 degrees C: deltaH=31.5 kcal mol-1; deltaS=105 cal deg-1 mol-1; and deltaCp=5215 cal deg-1 mol-1.     

Stability and physicochemical properties of the bovine brain phosphatidylethanolamine-binding protein.

The equilibrium behaviour of the bovine phosphatidylethanolamine-binding protein (PEBP) has been studied under various conditions of pH, temperature and urea concentration. Far-UV and near-UV CD, fluorescence and Fourier transform infrared spectroscopies indicate that, in its native state, PEBP is mainly composed of beta-sheets, with Trp residues mostly localized in a hydrophobic environment; these results suggest that the conformation of PEBP in solution is similar to the three-dimensional structure determined by X-ray crystallography. The pH-induced conformational changes show a transition midpoint at pH 3.0, implying nine protons in the transition. At neutral pH, the thermal denaturation is irreversible due to protein precipitation, whereas at acidic pH values the protein exhibits a reversible denaturation. The thermal denaturation curves, as monitored by CD, fluorescence and differential scanning calorimetry, support a two-state model for the equilibrium and display coincident values with a melting temperature Tm = 54 degrees C, an enthalpy change DeltaH = 119 kcal.mol-1 and a free energy change DeltaG(H2O, 25 degrees C) = 5 kcal.mol-1. The urea-induced unfolding profiles of PEBP show a midpoint of the two-state unfolding transition at 4.8 M denaturant, and the stability of PEBP is 4.5 kcal.mol-1 at 25 degrees C. Moreover, the surface active properties indicate that PEBP is essentially a hydrophilic protein which progressively unfolds at the air/water interface over the course of time. Together, these results suggest that PEBP is well-structured in solution but that its conformation is weakly stable and sensitive to hydrophobic conditions: the PEBP structure seems to be flexible and adaptable to its environment.     

Thermodynamic study of yeast phosphoglycerate kinase

Enthalpies of binding of MgADP, MgATP, and 3-phosphoglycerate to yeast phosphoglycerate kinase have been determined by flow calorimetry at 9.95-32.00 degrees C. Combination of these data with published dissociation constants [Scopes, R.K. (1978) Eur. J. Biochem. 91, 119-129] yielded the following thermodynamic parameters for the binding of 3-phosphoglycerate at 25 degrees C: delta Go = -6.76 +/- 0.11 kcal mol-1, delta H = 3.74 +/- 0.08 kcal mol-1, delta So = 35.2 +/- 0.6 cal K-1 mol-1, and delta Cp = 0.12 +/- 0.32 kcal K-1 mol-1. The thermal unfolding of phosphoglycerate kinase in the absence and presence of the ligands listed above was studied by differential scanning calorimetry. The temperature of half-completion, t 1/2, of the denaturation and the denaturational enthalpy are increased by the binding of the ligands, the increase in t 1/2 being a manifestation of Le Chatelier's principle and that in enthalpy reflecting the enthalpy of dissociation of the ligand. Only one denaturational peak was observed under all conditions, and in contrast with the case of yeast hexokinase [Takahashi, K., Casey, J.L., & Sturtevant, J.M. (1981) Biochemistry 20, 4693-4697], no definitive evidence for the unfolding of more than one domain was obtained.     

A new method for determining the heat capacity change for protein folding

In order to use results from calorimetry or thermal unfolding curves to estimate the free energy change for protein unfolding at 25 degrees C, it is necessary to know the change in heat capacity for unfolding, delta Cp. We describe a new method for measuring delta Cp which is based on results from urea and thermal unfolding curves but does not require a calorimeter. We find that delta Cp = 1650 +/- 200 cal/(deg.mol) for the unfolding of ribonuclease T1 and that delta Cp = 2200 +/- 300 cal/(deg.mol) for the unfolding of ribonuclease A.     

Thermodynamics of unfolding of beta-trypsin at pH 2.8

The unfolding equilibrium of beta-trypsin induced by thermal and chemical denaturation was thermodynamically characterized. Thermal unfolding equilibria were monitored using UV absorption and both far- and near-UV CD spectroscopy, while fluorescence was used to monitor urea-induced transitions. Thermal and urea transition curves are reversible and cooperative and both sets of data can be reasonably fitted using a two-state model for the unfolding of this protein. Plots of the fraction denatured, calculated from thermal denaturation curves at different wavelengths, versus temperature are coincident. In addition, the ratio of the enthalpy of denaturation obtained by scanning calorimetry to the van't Hoff enthalpy is close to unity, which supports the two-state model. Considering the differences in experimental approaches, the value for the stability of beta-trypsin estimated from spectroscopic data (deltaGu = 6.0 +/- 0.2 kcal/mol) is in reasonable agreement with the value calculated from urea titration curves (deltaGUH2O = 5.5 +/- 0.3 kcal/mol) at pH 2.8 and 300 degrees K.     

Calorimetric study of tryptophan synthase alpha-subunit and two mutant proteins

Heat-denaturation of tryptophan synthase alpha-subunit from E. coli and two mutant proteins (Glu 49 leads to Gln or Ser; called Gln 49 or Ser 49, respectively) has been studied by the scanning microcalorimetric method at various pH, in an attempt to elucidate the role of individual amino acid residues in the conformational stability of a protein. The partial specific heat capacity in the native state at 20 degrees, Cp20, has been found to be (0.43 +/- 0.02) cal . k-1 . g-1, the unfolding heat capacity change, delta dCp, (0.10 +/- 0.01) cal . K-1 . g-1, and the unfolding enthalpy value extrapolated to 110 degrees, delta dh110, (9.3 +/- 0.5) cal . g-1 for the three proteins. The value of Cp20 was larger than those found for "fully compact protein" and that of delta dh110 was smaller. Unfolding Gibbs energy, delta dG at 25 degrees for Wild-type, Gln 49, and Ser 49 were 5.8, 8.4, and 7.1 kcal . mol-1 at pH 9.3, respectively. Unfolding enthalpy, delta dH, of the three proteins seemed to be the same and equal to (23.2 +/- 1.2) kcal . mol-1 at 25 degrees. As a consequence of the same value of delta dH and the different value in delta dG, substantial differences in unfolding entropy, delta dS, were found for the three proteins. The values of delta dG for the three proteins at 25 degrees coincided with those from equilibrium methods of denaturation by guanidine hydrochloride.     

Unfolding of trp repressor studied using fluorescence spectroscopic techniques.

The unfolding properties of the trp repressor of Escherichia coli have been studied using a number of different time-resolved and steady-state fluorescence approaches. Denaturation by urea was monitored by the average fluorescence emission energy of the intrinsic tryptophan residues of the repressor. These data were consistent with a two-state transition from dimer to unfolded monomer with a free energy of unfolding of 19.2 kcal/mol. The frequency response profiles of the fluorescence emission brought to light subtle urea-induced modifications of the intrinsic tryptophan decay parameters both preceding and following the main unfolding transition. The increase of lifetime induced by urea required higher concentrations of urea than the increase in the total intensity described by Gittelman and Matthews [(1990) Biochemistry 29, 7011]. This indicates that the intensity increase has both dynamic and static origins. To assess the effect of tryptophan binding upon repressor stability, and to determine whether repressor oligomerization would be detectable in an unfolding experiment, we examined denaturation profiles of repressor labeled with the long-lived fluorescence probe 5-(dimethylamino)naphthalene-1-sulfonyl (DNS), by monitoring the average rotational correlation time of the probe. These experiments revealed a protein concentration dependent transition at low urea concentrations. This transition was promoted by tryptophan binding. We ascribe this transition to urea-induced dissociation of repressor tetramers. The main unfolding transition of the dimer to unfolded monomer was also observable using this technique, and the free energies associated with this transition were 18.3 kcal/mol in the absence of tryptophan and 24.1 kcal/mol in its presence, demonstrating that co-repressor binding stabilizes the repressor dimer against denaturation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermal denaturation of iso-1-cytochrome c variants: comparison with solvent denaturation.

Thermal denaturation studies as a function of pH were carried out on wild-type iso-1-cytochrome c and three variants of this protein at the solvent-exposed position 73 of the sequence. By examining the enthalpy and Tm at various pH values, the heat capacity increment (delta Cp), which is dominated by the degree of change in nonpolar hydration upon protein unfolding, was found for the wild type where lysine 73 is normally present and for three variants. For the Trp 73 variant, the delta Cp value (1.15 +/- 0.17 kcal/mol K) decreased slightly relative to wild-type iso-1-cytochrome c (1.40 +/- 0.06 kcal/mol K), while for the Ile 73 (1.65 +/- 0.07 kcal/mol K) and the Val 73 (1.50 +/- 0.06 kcal/mol K) variants, delta Cp increased slightly. In previous studies, the Trp 73, Ile 73, and Val 73 variants have been shown to have decreased m-values in guanidine hydrochloride denaturations relative to the wild-type protein (Hermann L, Bowler BE, Dong A, Caughey WS. 1995. The effects of hydrophilic to hydrophobic surface mutations on the denatured state of iso-1-cytochrome c: Investigation of aliphatic residues. Biochemistry 34:3040-3047). Both the m-value and delta Cp are related to the change in solvent exposure upon unfolding and other investigators have shown a correlation exists between these two parameters. However, for this subset of variants of iso-1-cytochrome c, a lack of correlation exists which implies that there may be basic differences between the guanidine hydrochloride and thermal denaturations of this protein. Spectroscopic data are consistent with different denatured states for thermal and guanidine hydrochloride unfolding. The different response of m-values and delta Cp for these variants will be discussed in this context.     

Protein stability: functional dependence of denaturational Gibbs energy on urea concentration

Determination of protein stability (DeltaGD0) from the conformational transition curve induced by a chemical denaturant is problematic; for different values of DeltaGD0, the value of the Gibbs energy change on denaturation (DeltaGD) in the absence of the denaturant are obtained when different extrapolation methods are used to analyze the same set of (DeltaGD, denaturant concentration) data [Pace, C. N. (1986) Methods Enzymol. 131, 266-280]. We propose a practical solution to this problem and use it to test the dependence of DeltaGD of lysozyme, ribonuclease-A, and cytochrome-c on [urea], the molar urea concentration. This method employs (i) measurements of the urea-induced denaturation in the presence of different guanidine hydrochloride (GdnHCl) concentrations which by themselves disrupt the native state of the protein at the same temperature and pH at which denaturations by urea and GdnHCl have been measured; (ii) estimation of DeltaGDcor, the value of DeltaGD corrected for the effect of GdnHCl on the urea-induced denaturation using the relation (DeltaGDcor = DeltaGD + mg [GdnHCl] = DeltaGD0 - mu [urea], where mg and mu are the dependencies of DeltaGD on [GdnHCl] and [urea], respectively) whose parameters are all determined from experimental denaturation data; and (iii) mapping of DeltaGDcor onto the DeltaGD versus [urea] plot obtained in the absence of GdnHCl. Our results convincingly show that (i) [urea] dependence of DeltaGD of each protein is linear over the full concentration range; (ii) the effect of urea and GdnHCl on protein denaturation is additive; and (iii) KCl affects the urea-induced denaturation if the native protein contains charge-charge interaction and/or anion binding site, in a manner which is consistent with the crystal structure data.     

Thermodynamics of ribonuclease T1 denaturation.

Differential scanning calorimetry has been used to investigate the thermodynamics of denaturation of ribonuclease T1 as a function of pH over the pH range 2-10, and as a function of NaCl and MgCl2 concentration. At pH 7 in 30 mM PIPES buffer, the thermodynamic parameters are as follows: melting temperature, T1/2 = 48.9 +/- 0.1 degrees C; enthalpy change, delta H = 95.5 +/- 0.9 kcal mol-1; heat capacity change, delta Cp = 1.59 kcal mol-1 K-1; free energy change at 25 degrees C, delta G degrees (25 degrees C) = 5.6 kcal mol-1. Both T1/2 = 56.5 degrees C and delta H = 106.1 kcal mol-1 are maximal near pH 5. The conformational stability of ribonuclease T1 is increased by 3.0 kcal/mol in the presence of 0.6 M NaCl or 0.3 M MgCl2. This stabilization results mainly from the preferential binding of cations to the folded conformation of the protein. The estimates of the conformational stability of ribonuclease T1 from differential scanning calorimetry are shown to be in remarkably good agreement with estimates derived from an analysis of urea denaturation curves.     

Guanidinium chloride- and urea-induced unfolding of the dimeric enzyme glucose oxidase

We have carried out a systematic study on the guanidinium chloride- and urea-induced unfolding of glucose oxidase from Aspergillus niger, an acidic dimeric enzyme, using various optical spectroscopic techniques, enzymatic activity measurements, glutaraldehyde cross-linking, and differential scanning calorimetry. The urea-induced unfolding of GOD was a two-state process with dissociation and unfolding of the native dimeric enzyme molecule occurring in a single step. On the contrary, the GdmCl-induced unfolding of GOD was a multiphasic process with stabilization of a conformation more compact than the native enzyme at low GdmCl concentrations and dissociation along with unfolding of enzyme at higher concentrations of GdmCl. The GdmCl-stabilized compact dimeric intermediate of GOD showed an enhanced stability against thermal and urea denaturation as compared to the native GOD dimer. Comparative studies on GOD using GdmCl and NaCl demonstrated that binding of the Gdm(+) cation to the enzyme results in stabilization of the compact dimeric intermediate of the enzyme at low GdmCl concentrations. An interesting observation was that a slight difference in the concentration of urea and GdmCl associated with the unfolding of GOD was observed, which is in violation of the 2-fold rule for urea and GdmCl denaturation of proteins. This is the first report where violation of the 2-fold rule has been observed for a multimeric protein.     

pH dependence of the stability of barstar to chemical and thermal denaturation.

Equilibrium unfolding of barstar with guanidine hydrochloride (GdnHCl) and urea as denaturants as well as thermal unfolding have been carried out as a function of pH using fluorescence, far-UV and near-UV CD, and absorbance as probes. Both GdnHCl-induced and urea-induced denaturation studies at pH 7 show that barstar unfolds through a two-state F<->U mechanism and yields identical values for delta GU, the free energy difference between the fully folded (F) and unfolded (U) forms, of 5.0 +/- 0.5 kcal.mol-1 at 25 degrees C. Thermal denaturation of barstar also follows a two-state F<->U unfolding transition at pH 7, and the value of delta GU at 25 degrees C is similar to that obtained from chemical denaturation. The pH dependence of denaturation by GdnHCl is complex. The Cm value (midpoint of the unfolding transition) has been used as an index for stability in the pH range 2-10, because barstar does not unfold through a two-state transition on denaturation by GdnHCl at all pH values studied. Stability is maximum at pH 2-3, where barstar exists in a molten globule-like form that forms a large soluble oligomer. The stability decreases with an increase in pH to 5, the isoelectric pH of the protein. Above pH 5, the stability increases as the pH is raised to 7. Above pH 8, it again decreases as the pH is raised to 10. The decrease in stability from pH 7 to 5 in wild-type (wt) barstar, which is shown to be characterized by an apparent pKa of 6.2 +/- 0.2, is not observed in H17Q, a His 17-->Gln 17 mutant form of barstar. This decrease in stability has therefore been correlated with the protonation of His 17 in barstar. The decrease in stability beyond pH 8 in wt barstar, which is characterized by an apparent pKa of 9.2 +/- 0.2, is not detected in BSCCAA, the Cys 40 Cys 82-->Ala 40 Ala 82 double mutant form of barstar. Thus, this decrease in stability has been correlated with the deprotonation of at least one of the two cysteines present in wt barstar. The increase in stability from pH 5 to 3 is characterized by an apparent pKa of 4.6 +/- 0.2 for wt barstar and BSCCAA, which is similar to the apparent pKa that characterizes the structural transition leading to the formation of the A form. The use of Cm as an index of stability has been supported by thermal denaturation studies.(ABSTRACT TRUNCATED AT 400 WORDS)     

A thermodynamic analysis of a family of small globular proteins: SH3 domains.

The stability and folding thermodynamics of two SH3-domains, belonging to Fyn and Abl proteins, have been studied by scanning calorimetry and urea-induced unfolding. They undergo an essentially two-state unfolding with parameters similar to those of the previously studied alpha-spectrin SH3 domain. The correlations between the thermodynamic parameters (heat capacity increment, delta Cp,U, the proportionality factor, m, and the Gibbs energy, delta Gw298) of unfolding and some integral structural parameters, such as polar and non-polar areas exposed upon domain denaturation, have been analyzed. The experimental data on delta Cp,U and the m-factor of the linear extrapolation model (LEM) obey the simple empirical correlations deduced elsewhere. The Gibbs energies calculated from the DSC data were compared with those found by fitting urea-unfolding curves to the LEM and the denaturant-binding model (DBM). The delta Gw298 values found with DBM correlate better with the DSC data, while those obtained with LEM are systematically smaller. The systematic difference between the parameters calculated with LEM and DBM are explained by an inherent imperfection of the LEM.     

DSC studies of the conformational stability of barstar wild-type.

The temperature induced unfolding of barstar wild-type of bacillus amyloliquefaciens (90 residues) has been characterized by differential scanning microcalorimetry. The process has been found to be reversible in the pH range from 6.4 to 8.3 in the absence of oxygen. It has been clearly shown by a ratio of delta HvH/delta Hcal near 1 that denaturation follows a two-state mechanism. For comparison, the C82A mutant was also studied. This mutant exhibits similar reversibility, but has a slightly lower transition temperature. The transition enthalpy of barstar wt (303 kJ mol-1) exceeds that of the C82A mutant (276 kJ mol-1) by approximately 10%. The heat capacity changes show a similar difference, delta Cp being 5.3 +/- 1 kJ mol-1 K-1 for the wild-type and 3.6 +/- 1 kJ mol-1 K-1 for the C82A mutant. The extrapolated stability parameters at 25 degrees C are delta G0 = 23.5 +/- 2 kJ mol-1 for barstar wt and delta G0 = 25.5 +/- 2 kJ mol-1 for the C82A mutant.     

Erythropoietin unfolding: thermodynamics and its correlation with structural features

Human erythropoietin (EPO) is a glycoprotein hormone considered to be the principal regulator of red blood cell formation. Although its recombinant version (rEPO) has been widely used for treatment of various anemias and its biological effects are relatively well-known, we know little about its biophysical properties and their relation to its structure. To gain a fuller understanding of the structural and functional properties of rEPO on the molecular level we followed its thermal and urea-induced unfolding at different pH (3.1-9.4) and urea concentrations (0-8 M) using spectropolarimetry, UV absorption, intrinsic emission fluorescence, and differential scanning calorimetry. Our results show that under a variety of conditions rEPO undergoes thermal or urea-induced denaturation that may be considered as a reversible two-state process characterized by unusually high (thermal) or moderate (urea-induced) extent of the residual structure. The highest thermal stability of the protein observed in aqueous solutions at physiological pH appears to be due to the largest difference in the extent of structure in the denatured and native state at this pH. The comparison between experimentally determined energetics of rEPO denaturation and its structure-based calculations indicates that the parametrization of thermodynamic quantities in terms of changes in solvent accessible nonpolar and polar surface areas resulting from protein unfolding can be successfully used provided that these changes are estimated from combination of experimentally determined deltaC(o)p and deltaH(o) values and not calculated from the structure of the protein's folded and assumingly fully unfolded state.     

Stability of yeast iso-1-ferricytochrome c as a function of pH and temperature.

Absorbance-detected thermal denaturation studies of the C102T variant of Saccharomyces cerevisiae iso-1-ferricytochrome c were performed between pH 3 and 5. Thermal denaturation in this pH range is reversible, shows no concentration dependence, and is consistent with a 2-state model. Values for free energy (delta GD), enthalpy (delta HD), and entropy (delta SD) of denaturation were determined as functions of pH and temperature. The value of delta GD at 300 K, pH 4.6, is 5.1 +/- 0.3 kcal mol-1. The change in molar heat capacity upon denaturation (delta Cp), determined by the temperature dependence of delta HD as a function of pH (1.37 +/- 0.06 kcal mol-1 K-1), agrees with the value determined by differential scanning calorimetry. pH-dependent changes in the Soret region indicate that a group or groups in the heme environment of the denatured protein, probably 1 or both heme propionates, ionize with a pK near 4. The C102T variant exhibits both enthalpy and entropy convergence with a delta HD of 1.30 kcal mol-1 residue-1 at 373.6 K and a delta SD of 4.24 cal mol-1 K-1 residue-1 at 385.2 K. These values agree with those for other single-domain, globular proteins.