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.     

Scan-rate dependence in protein calorimetry: the reversible transitions of Bacillus circulans xylanase and a disulfide-bridge mutant.

The stabilities of Bacillus circulans xylanase and a disulfide-bridge-containing mutant (S100C/N148C) were investigated by differential scanning calorimetry (DSC) and thermal inactivation kinetics. The thermal denaturation of both proteins was found to be irreversible, and the apparent transition temperatures showed a considerable dependence upon scanning rate. In the presence of low (nondenaturing) concentrations of urea, calorimetric transitions were observed for both proteins in the second heating cycle, indicating reversible denaturation occurs under those conditions. However, even for these reversible processes, the DSC curves for the wild-type protein showed a scan-rate dependence that was similar to that in the absence of urea. Calorimetric thermograms for the disulfide mutant were significantly less scan-rate dependent in the presence of urea than in the urea-free buffer. The present data show that, just as for irreversible transitions, the apparent transition temperature for the reversible denaturation of proteins can be scan-rate dependent, confirming the prediction of Lepock et al. (Lepock JR, Rithcie KP, Kolios MC, Rodahl AM, Heinz KA, Kruuf J, 1992, Biochemistry 31:12706-12712). The kinetic factors responsible for scan-rate dependence may lead to significant distortions and asymmetry of endotherms, especially at higher scanning rates. This points to the need to check for scan-rate dependence, even in the case of reversible denaturation, before any attempt is made to analyze asymmetric DSC curves by standard thermodynamic procedures. Experiments with the disulfide-bridge-containing mutant indicate that the introduction of the disulfide bond provides additional stabilization of xylanase by changing the rate-limiting step on the thermal denaturation pathway.     

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)     

Thermal analysis of CHL V79 cells using differential scanning calorimetry: implications for hyperthermic cell killing and the heat shock response

Differential scanning calorimetry (DSC) was used to assay thermal transitions that might be responsible for cell death and other responses to hyperthermia or heat shock, such as induction of heat shock proteins (HSP), in whole Chinese hamster lung V79 cells. Seven distinct peaks, six of which are irreversible, with transition temperatures from 49.5 degrees C to 98.9 degrees C are detectable. These primarily represent protein denaturation with minor contributions from DNA and RNA melting. The onset temperature of denaturation, 38.7 degrees C, is shifted to higher temperatures by prior heat shock at 43 degrees and 45 degrees C, indicative of irreversible denaturation occurring at these temperatures. Thus, using DSC it is possible to demonstrate significant denaturation in a mammalian cell line at temperatures and times of exposure sufficient to induce hyperthermic damage and HSP synthesis. A model was developed based on the assumption that the rate limiting step of hyperthermic cell killing is the denaturation of a critical target. A transition temperature of 46.3 degrees C is predicted for the critical target in V79 cells. No distinct transition is detectable by DSC at this temperature, implying that the critical target comprises a small fraction of total denaturable material. The short chain alcohols methanol, ethanol, isopropanol, and t-butanol are known hyperthermic sensitizers and ethanol is an inducer of HSP synthesis. These compounds non-specifically lower the denaturation temperature of cellular protein. Glycerol, a hyperthermic protector, non-specifically raises the denaturation temperature for proteins denaturing below 60 degrees C. Thus, there is a correlation between the effect of these compounds on protein denaturation in vivo and their effect on cellular sensitivity to hyperthermia.     

Thermal denaturation of the Ca2(+)-ATPase of sarcoplasmic reticulum reveals two thermodynamically independent domains

Inactivation of Ca2+ uptake and ATPase activity of the Ca2(+)-ATPase of rabbit sarcoplasmic reticulum was measured and compared to the thermal denaturation of the enzyme as measured by differential scanning calorimetry (DSC) and fluorescence spectroscopy. Two fluorophores were monitored: intrinsic tryptophan (localized in the transmembrane region) and fluorescein isothiocyanate (FITC)-labeled Lys-515 (located in the nucleotide binding domain). Inactivation, defined as loss of activity, and denaturation, defined as conformational unfolding, were irreversible under the conditions used. Activation energies (EA) and frequency factors (A) for inactivation were obtained for the enzyme in 1 mM EGTA and 1 mM Ca2+. These were transformed to a transition temperature for inactivation, Tm (defined as the temperature of half-inactivation when temperature is scanned upward at 1 degree C/min). All denaturation profiles were fit with an irreversible model to obtain EA and Tm for each transition, and the values of these parameters for denaturation were compared to the values for inactivation. In EGTA, denaturation obeys a single-step model (Tm = 49 degrees C), but a two-step model is required to fit the DSC provile of the enzyme in 1 mM Ca2+. The specific locations of tryptophan and the fluorescein label were used to demonstrate that denaturation in Ca2+ occurs through two distinct thermodynamic domains. Domain I (Tm = 50 degrees C) consists of the nucleotide binding region and most likely the phosphorylation and transduction regions [MacLennan, D. H., Brandl, C. J., Korczak, B., & Green, N. M. (1985) Nature 316, 696-700].(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of the thermal unfolding of porcine procarboxypeptidase A and its functional pieces by differential scanning calorimetry

Porcine pancreatic procarboxypeptidase A and its tryptic peptides, carboxypeptidase A and the activation segment, have been studied by high-sensitivity differential scanning calorimetry (DSC). The thermal denaturation of the zymogen and the active enzyme has been carried out at two pH values, 7.5 and 9.0, at different ionic strengths and at different scan rates. The endothermic transitions for these two proteins were always irreversible under all conditions investigated. The denaturation behaviour of both proteins seems to fit very well with the kinetic model for the DSC study of irreversible unfolding of proteins recently proposed by one of our groups. From this model, the activation energies obtained for the denaturation of the pro- and carboxypeptidase were 300 +/- 20 kJ mol-1 and 250 +/- 14 kJ mol-1 respectively. On the other hand, the isolated activation segment appears as a thermostable piece with a highly reversible thermal unfolding which follows a two-state process. The denaturation temperature observed for the isolated segment was always at least 15 K higher than those of the zymogen and the active enzyme.     

Thermal conformational changes of bovine fibrinogen by differential scanning calorimetry and circular dichroism.

The thermal denaturation of bovine fibrinogen has been investigated using differential scanning calorimetry (DSC) and circular dichroism (CD) spectroscopy. Differential scanning calorimetry measurements were carried out while changing the scan-rate. The transition at 57 degrees C was found to be irreversible and highly scan-rate dependent, suggesting that the denaturation is, at least in part, under kinetic control. The secondary structural changes at various temperatures were monitored by far-ultraviolet CD spectroscopy. These results show that the DSC transition for the thermal denaturation of bovine fibrinogen can be interpreted in terms of a kinetic process, N --> F, where k is a first-order kinetic constant that changes with temperature according to the Arrhenius equation. An important transition peak was observed at 78.8 degrees C which is attributed to the C-terminal parts of the Aalpha chains of fibrinogen.     

Heat-induced conversion of beta(2)-microglobulin and hen egg-white lysozyme into amyloid fibrils

Thermodynamic parameters characterizing protein stability can be obtained for a fully reversible folding/unfolding system directly by differential scanning calorimetry (DSC). However, the reversible DSC profile can be altered by an irreversible step causing aggregation. Here, to obtain insight into amyloid fibrils, ordered and fibrillar aggregates responsible for various amyloidoses, we studied the effects on human beta(2)-microglobulin and hen egg-white lysozyme of a combination of agitation and heating. Aggregates formed by mildly agitating protein solutions in the native state in the presence of NaCl were heated in the cell of the DSC instrument. For beta(2)-microglobulin, with an increase in the concentration of NaCl at neutral pH, the thermogram began to show an exothermic transition accompanied by a large decrease in heat capacity, followed by a kinetically controlled thermal response. Similarly, the aggregated lysozyme at a high concentration of NaCl revealed a similar distinct transition in the DSC thermogram over a wide pH range. Electron microscopy demonstrated the conformational change into amyloid fibrils. Taken together, the combined use of agitation and heating is a powerful way to generate amyloid fibrils from two proteins, beta(2)-microglobulin and hen egg-white lysozyme, and to evaluate the effects of heat on fibrillation, in which the heat capacity is crucial to characterizing the transition.     

Irreversible Denaturation of Maltodextrin Glucosidase Studied by Differential Scanning Calorimetry,Circular Dichroism,and Turbidity Measurements

Thermal denaturation of Escherichia coli maltodextrin glucosidase was studied by differential scanning calorimetry, circular dichroism (230 nm), and UV-absorption measurements (340 nm), which were respectively used to monitor heat absorption, conformational unfolding, and the production of solution turbidity. The denaturation was irreversible, and the thermal transition recorded at scan rates of 0.5–1.5 K/min was significantly scan-rate dependent, indicating that the thermal denaturation was kinetically controlled. The absence of a protein-concentration effect on the thermal transition indicated that the denaturation was rate-limited by a mono-molecular process. From the analysis of the calorimetric thermograms, a one-step irreversible model well represented the thermal denaturation of the protein. The calorimetrically observed thermal transitions showed excellent coincidence with the turbidity transitions monitored by UV-absorption as well as with the unfolding transitions monitored by circular dichroism. The thermal denaturation of the protein was thus rate-limited by conformational unfolding, which was followed by a rapid irreversible formation of aggregates that produced the solution turbidity. It is thus important to note that the absence of the protein-concentration effect on the irreversible thermal denaturation does not necessarily means the absence of protein aggregation itself. The turbidity measurements together with differential scanning calorimetry in the irreversible thermal denaturation of the protein provided a very effective approach for understanding the mechanisms of the irreversible denaturation. The Arrhenius-equation parameters obtained from analysis of the thermal denaturation were compared with those of other proteins that have been reported to show the one-step irreversible thermal denaturation. Maltodextrin glucosidase had sufficiently high kinetic stability with a half-life of 68 days at a physiological temperature (37°C).     

L-phenylalanine binding and domain organization in human phenylalanine hydroxylase: a differential scanning calorimetry study

Human phenylalanine hydroxylase (hPAH) is a tetrameric enzyme that catalyzes the hydroxylation of L-phenylalanine (L-Phe) to L-tyrosine; a dysfunction of this enzyme causes phenylketonuria. Each subunit in hPAH contains an N-terminal regulatory domain (Ser2-Ser110), a catalytic domain (Asp112-Arg410), and an oligomerization domain (Ser411-Lys452) including dimerization and tetramerization motifs. Two partially overlapping transitions are seen in differential scanning calorimetry (DSC) thermograms for wild-type hPAH in 0.1 M Na-Hepes buffer, 0.1 M NaCl, pH 7.0. Although these transitions are irreversible, studies on their scan-rate dependence support that the equilibrium thermodynamics analysis is permissible in this case. Comparison with the DSC thermograms for truncated forms of the enzyme, studies on the protein and L-Phe concentration effects on the transitions, and structure-energetic calculations based on a modeled structure support that the thermal denaturation of hPAH occurs in three stages: (i) unfolding of the four regulatory domains, which is responsible for the low-temperature calorimetric transition; (ii) unfolding of two (out of the four) catalytic domains, which is responsible for the high-temperature transition; and (iii) irreversible protein denaturation, which is likely responsible for the observed exothermic distortion in the high-temperature side of the high-temperature transition. Stages 1 and 2 do not appear to be two-state processes. We present an approach to the analysis of ligand effects on DSC transition temperatures, which is based on the general binding polynomial formalism and is not restricted to two-state transitions. Application of this approach to the L-Phe effect on the DSC thermograms for hPAH suggests that (i) there are no binding sites for L-Phe in the regulatory domains; therefore, contrary to the common belief, the activation of PAH by L-Phe seems to be the result of its homotropic cooperative binding to the active sites. (ii) The regulatory domain appears to be involved in cooperativity through its interactions with the catalytic and oligomerization domains; thus, upon regulatory domain unfolding, the cooperativity in the binding of L-Phe to the catalytic domains seems to be lost and the value of the L-Phe concentration corresponding to half-saturation is increased. Overall, our results contribute to the understanding of the conformational stability and the substrate-induced cooperative activation of this important enzyme.     

Three easy pieces

BackgroundDifferential scanning calorimetry is a powerful method that provides a complete thermodynamic characterization of the stability of a protein as a function of temperature. There are, however, circumstances that preclude a complete analysis of DSC data. The most common ones are irreversible denaturation transitions or transitions that take place at temperatures that are beyond the temperature limit of the instrument. Even for a protein that undergoes reversible thermal denaturation, the extrapolation of the thermodynamic data to lower temperatures, usually 25 °C, may become unreliable due to difficulties in the determination of ΔC_p.MethodsThe combination of differential scanning calorimetry and isothermal chemical denaturation allows reliable thermodynamic analysis of protein stability under less than ideal conditions.Results and conclusionsThis paper demonstrates how DSC can be used in combination with chemical denaturation to address three different scenarios: 1) estimation of an accurate ΔC_p value for a reversible denaturation using as a test system the envelope HIV-1 glycoprotein gp120; 2) determination of the Gibbs energy of stability in the region in which thermal denaturation is irreversible using HEW lysozyme at different pH values; and, 3) determination of Gibbs energy of stability for a thermostable protein, thermolysin. This article is part of a Special Issue entitled Microcalorimetry in the BioSciences — Principles and Applications, edited by Fadi Bou-Abdallah.     

The Kinetic Stability of Cytochrome c Oxidase: Effect of Bound Phospholipid and Dimerization

Thermally induced transitions of the 13-subunit integral membrane protein bovine cytochrome c oxidase (CcO) have been studied by differential scanning calorimetry (DSC) and circular dichroism (CD). Thermal denaturation of dodecyl maltoside solubilized CcO proceeds in two consecutive, irreversible, kinetically driven steps with the apparent transition temperatures at ∼ 51°C and ∼ 61°C (5μM CcO at scan rate of 1.5 K/min). The thermal denaturation data were analyzed according to the Lyubarev and Kurganov model of two consecutive irreversible steps. However, because of the limitation of the model to describe the complex mechanism of the thermal denaturation of CcO, the obtained results were utilized only for comparison purposes of kinetic stabilities of CcO under specific protein concentration (5μM) and scan rate (1.5 K/min). This enabled us to show that both the amphiphilic environment and the self-association state of CcO affect its kinetic stability. Kinetic stabilities of both steps are significantly decreased when all of the phospholipids are removed from CcO by phospholipase A₂ (the half-life decreases at 37°C). Conversely, dimerization of CcO induced by sodium cholate significantly increases its kinetic stability of only the first step (the half-life increases at 37°C). Protein concentration-dependent nonspecific oligomerization also indicate mild stabilization of CcO. Both, reversed-phase high-performance liquid chromatography (HPLC) and SDS-PAGE subunit analysis reveal that the first step of thermal denaturation involves dissociation of subunits III, VIa, VIb, and VIIa, whereas the second step is less well defined and most likely involves global unfold and aggregation of the remaining subunits. Electron transport activity of CcO decreases in a sigmoidal manner during the first transition and this dependence is very well described by kinetic parameters for the first step of the thermal transition. Therefore, dissociation of subunit III and/or VIIa is responsible for temperature-induced inactivation of CcO because VIa and VIb can be removed from CcO without affecting the enzyme activity. These results demonstrate an important role of tightly bound phospholipids and oligomeric state (particularly the dimeric form) of CcO for kinetic stability of the protein.     

Effects of metal ligation and oxygen on the reversibility of the thermal denaturation of Pseudomonas aeruginosa azurin.

Thermodynamic equilibrium transition models in DSC are only applicable to reversible processes, but reversibility of the thermal transitions of proteins is comparatively rare because of intermolecular aggregation of denatured proteins and the degradation that occurs at high temperatures. The cupredoxin azurin from Pseudomonas aeruginosa has previously been found to exhibit irreversible thermal denaturation, both as holo- and apoprotein [Engeseth, H. R., and McMillin, D. R. (1986) Biochemistry 25, 2448-2455]. In this study, however, we demonstrate that this beta-barrel protein of Greek key topology in fact unfolds reversibly in anaerobic solutions when nonreducible metal ions are ligated to the protein. We show that it is the metal-coordinating cysteine residue (C112) that becomes exclusively oxidized in a transition metal catalyzed oxidation reaction with dissolved O(2) at high temperatures. Both Cu(I)- and Zn(II)-coordinating wild-type azurin therefore unfold reversibly in anaerobic solutions, as well as the Zn(II)-coordinating disulfide-deficient C3A/C26A mutant. Correspondingly, apoazurin mutants C112A and C112S unfold reversibly, even in aerobic solutions, and exhibit nearly perfect two-state transitions. Unfolding of Cu(II)-coordinating azurin is, on the other hand, always irreversible due to autoxidation of the thiolate resulting in Cu(I) and a thiyl radical prone to oxidation.     

Three steps in the thermal unfolding of F-actin: an experimental evidence

The development of differential scanning calorimetry has resulted in an increased interest in studies of the unfolding process in proteins with the aim of identifying domains and interactions with ligands or other proteins. Several of these studies were done with actin and showed that the thermal unfolding of F-actin occurs in at least three steps; this was interpreted as the denaturation of independent domains. In the present work, we have followed the thermal unfolding of F-actin using differential scanning calorimetry (DSC), CD spectroscopy, and probe fluorescence. We found that the three steps revealed through DSC are not the denaturation of independent domains. These three steps are a change in the environment of cys 374 at 49.5 degrees C; a modification at the nucleotide-binding site at 55 degrees C; and the unfolding of the peptide chain at 64 degrees C. Previous interpretations of the thermograms of F-actin were thus erroneous. Since DSC is now widely used to study proteins, our experimental approach and conclusions may also be relevant in denaturation studies of proteins in general.     

Stability parameters for one-step mechanism of irreversible protein denaturation: a method based on nonlinear regression of calorimetric peaks with nonzero deltaCp

Thermal transitions of many proteins have been found to be calorimetrically irreversible and scan-rate dependent. Calorimetric determinations of stability parameters of proteins which unfold irreversibly according to a first-order kinetic scheme have been reported. These methods require the approximation that the increase in heat capacity upon denaturation deltaCp is zero. A method to obtain thermodynamic parameters and activation energy for the two-state irreversible process N --> D from nonlinear fitting to calorimetric traces is proposed here. It is based on a molar excess heat capacity function which considers irreversibility and a nonzero constant deltaCp. This function has four parameters: (1) temperature at which the calorimetric profile reaches its maximal value (Tm), (2) calorimetric enthalpy at Tm (deltaHm), (3) deltaCp, and (4) activation energy (E). The thermal irreversible denaturation of subtilisin BPN' from Bacillus amyloliquefaciens was studied by differential scanning calorimetry at pH 7.5 to test our model. Transitions were found to be strongly scanning-rate dependent with a mean deltaCp value of 5.7 kcal K(-1)mol(-1), in agreement with values estimated by accessible surface area and significantly higher than a previously reported value.     

Differential scanning calorimetric study of the thermal unfolding of beta-lactamase I from Bacillus cereus.

The irreversible thermal unfolding of the class A beta-lactamase I from Bacillus cereus has been investigated at pH 7.0, using differential scanning calorimetry (DSC) and inactivation kinetic techniques. DSC transitions showed a single peak with a denaturation enthalpy of 646 kJ.mol-1 and were moderately scan rate dependent, suggesting that the process was partially kinetically controlled. The inactivation kinetics at constant temperature showed that the irreversible denaturation of the enzyme occurs as the sum of two exponential terms whose amplitudes are strongly temperature dependent within the transition range so that, at the lowest temperatures within this interval, irreversible inactivation would proceed mainly through the slow phase. The fraction of irreversibly denatured enzyme (D) as a function of temperature for a given scanning rate was calculated by numerical integration of the kinetic equation with temperature, using previously determined kinetic parameters. This D form was the most populated of the unfolded states only at temperatures well above the maximum in the calorimetric transition. Combination of the results of kinetic and DSC experiments has allowed us to separate the contribution of the final D state to the excess enthalpy change from the contribution arising from the reversibly denatured forms of the enzyme (I(i), i = 1,..., n), with the resulting conclusion that the scan rate dependence of the calorimetric traces was the result of two different dynamic effects, viz., the irreversible step and a slow relaxation process during formation of the reversibly denatured intermediate states. Finally, the problems of using results obtained at a single scan rate to validate the two-state kinetic model are commented on.     

Reversible and non-reversible thermal denaturation of lysozyme with varying pH at low ionic strength

DSC analysis has been used to quantify the reversibility of unfolding following thermal denaturation of lysozyme. Since the temperature at which protein unfolding occurs, T_m, varies with different solution conditions, the effect on the melting temperature and the degree of refolding after thermal denaturation in low ionic strength sodium phosphate buffers (5–1000 mM) over a range of pH (5–9) in the presence/absence of disaccharides is examined. This study compares the enthalpies of unfolding during successive heating cycles to quantify reversibility following thermal denaturation. The disaccharides, trehalose and maltose were used to assess if the disaccharide induced increase in T_m is reflected in the reversibility of thermally induced denaturation. There was extensive overlap between the T_m values where non-reversible and reversible thermal denaturation occurred. Indeed, for pH 6, at the highest and lowest T_m, no refolding was observed whereas refolding was observed for intermediate values, but with similar T_m values having different proportions of refolded protein. We established a method to measure the degree of reversible unfolding following thermal denaturation and hence indirectly, the degree to which protein is lost to irreversible aggregation, and show that solution conditions which increase melt transition temperatures do not automatically confer an increase in reversibility. This type of analysis may prove useful in assessing the stability of proteins in both the biopharmaceutical and food industries.     

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.     

Temperature stability of proteins: Analysis of irreversible denaturation using isothermal calorimetry

The structural stability of proteins has been traditionally studied under conditions in which the folding/unfolding reaction is reversible, since thermodynamic parameters can only be determined under these conditions. Achieving reversibility conditions in temperature stability experiments has often required performing the experiments at acidic pH or other nonphysiological solvent conditions. With the rapid development of protein drugs, the fastest growing segment in the pharmaceutical industry, the need to evaluate protein stability under formulation conditions has acquired renewed urgency. Under formulation conditions and the required high protein concentration (～100 mg/mL), protein denaturation is irreversible and frequently coupled to aggregation and precipitation. In this article, we examine the thermal denaturation of hen egg white lysozyme (HEWL) under irreversible conditions and concentrations up to 100 mg/mL using several techniques, especially isothermal calorimetry which has been used to measure the enthalpy and kinetics of the unfolding and aggregation/precipitation at 12°C below the transition temperature measured by DSC. At those temperatures the rate of irreversible protein denaturation and aggregation of HEWL is measured to be on the order of 1 day^?1. Isothermal calorimetry appears a suitable technique to identify buffer formulation conditions that maximize the long term stability of protein drugs.