Disagreements Between Calorimetric and Van't Hoff Enthalpies of Adsorption II: Effect of pH and pH Buffers on Phenobarbital Adsorption to Activated Carbon

The reported inconsistencies between the van't Hoff equation and calorimetry hinder the utility of thermodynamics in biochemical and pharmaceutical research. A novel thermodynamic approach is developed herein for ligand adsorption with a focus on the interpretation of calorimetric data in the presence of concurrent proton exchange reactions. Such exchange reactions typically result in a pH-dependence of calorimetric measurements that obscures intrinsic binding enthalpies. It is shown that for the adsorption of phenobarbital to activated carbon, the measured calorimetric enthalpy is a result of three linked acid/base equilibria. A model was established to predict the intrinsic binding enthalpy using 1) the adsorbate's pKa and 2) the adsorbate's enthalpy of protonation. The observed calorimetric enthalpy of binding exhibited both pH and buffer-dependence and was between -5 and -42 kJ/mol. Meanwhile, the predicted intrinsic enthalpy (-25.1 kJ/mol) of binding was in excellent agreement with the measured intrinsic enthalpy (-25.6 kJ/mol). Corrections to the observed calorimetric enthalpies allowed comparisons with enthalpies obtained from the van't Hoff method. It is shown that the predicted intrinsic calorimetric enthalpy agrees well with the van't Hoff enthalpies in instances where observed enthalpies significantly deviated. This treatment is general and is not specific to phenobarbital or activated carbon.     

Disagreements Between Calorimetric and Van't Hoff Enthalpies of Adsorption: A New Langmuir-like Model to Account for the Effect of Solvent Displacement Stoichiometry

The reported inconsistencies between calorimetry and the van't Hoff equation hinder the utility of thermodynamics in pharmaceutical research. In ligand binding or adsorption assays, it is believed that the van't Hoff equation falls short because of the lack of stoichiometric treatment in the equilibrium constant. A new modified Langmuir-Like equation that accounts for the stoichiometry of solute adsorption and solvent displacement is proposed in this work. The performance of the model was evaluated by studying the adsorption of phenobarbital from aqueous solutions by commercial activated carbon. The amount of water occupying the adsorption sites was estimated by graphical analysis of the ‘knee point’ of water-vapor adsorption isotherms and was found to correlate well with the relative percentage of hydroxyl and carbonyl surface groups. It was found that one phenobarbital molecule displaces 2-6 water molecules from the adsorption site. It is shown that adsorption enthalpy was not affected by the adjustment for stoichiometry, supporting the notion that the van't Hoff enthalpy is intrinsic and is independent of the stoichiometry of solvent displacement in Langmuir-based binding. The widely reported disparities between the van't Hoff and calorimetric enthalpies are unlikely to be from a stoichiometric origin.     

Structure characterization of human serum proteins in solution and dry state

Abstract: The present report describes application of advanced analytical methods to establish correlation between changes in human serum proteins of patients with coronary atherosclerosis (protein metabolism) before and after moderate beer consumption. Intrinsic fluorescence, circular dichroism (CD), differential scanning calorimetry and hydrophobicity (S_o) were used to study human serum proteins. Globulin and albumin from human serum (HSG and HSA, respectively) were denatured with 8 m urea as the maximal concentration. The results obtained provided evidence of differences in their secondary and tertiary structures. The thermal denaturation of HSA and HSG expressed in temperature of denaturation (T_d, °C), enthalpy (ΔH, kcal/mol) and entropy (ΔS kcal/mol K) showed qualitative changes in these protein fractions, which were characterized and compared with fluorescence and CD. Number of hydrogen bonds (n) ruptured during this process was calculated from these thermodynamic parameters and then used for determination of the degree of denaturation (%D). Unfolding of HSA and HSG fractions is a result of promoted interactions between exposed functional groups, which involve conformational changes of α‐helix, β‐sheet and aperiodic structure. Here evidence is provided that the loosening of the human serum protein structure takes place primarily in various concentrations of urea before and after beer consumption (BC). Differences in the fluorescence behavior of the proteins are attributed to disruption of the structure of proteins by denaturants as well as by the change in their compactability as a result of ethanol consumption. In summary, thermal denaturation parameters, fluorescence, S_o and the content of secondary structure have shown that HSG is more stable fraction than HSA.     

Stability of 11S globulin from Vicia faba seeds

Heat denaturation of 11S globulin, a dodecameric globular protein isolated from Vicia faba seeds was studied using scanning microcalorimetry at pH 7.6 and NaCl concentrations from 0 to 1 M. The specific enthalpy of denaturation was shown to be a linear function of temperature. The ratio of the calorimetric enthalpy to the effective one (Van't-Hoff's) per protomer of 11 S globulin was 0.9 ± 0.06. It is concluded that at first approximation 11 S globulin protomers denaturated independently in conformity with the two-state model. The plotted temperature-dependent specific free energy of 11 S globulin denaturation at different NaCl concentrations demonstrated that an increase in the salt content brought about the rise in protein stability. The maximum 11 S globulin stability is reached at about 300°K. The molar free energy of denaturation at 300°K in 1 M NaCl is 918 kJ/mol.     

Synthesis and stabilization of amino and carboxy terminal constrained collagenous peptides

Short collagenous peptides cross-linked at their amino and carboxy termini with Lys-Lys-dimer template(s) were synthesized, and the effect of the cross-linking on their stabilities was investigated by thermal denaturation experiments. Two chemoselective ligations were used for the construction of the amino and the carboxy cross-linked peptides. The thermal transition temperature (T_m) and the standard free energies (ΔG°) of the cross-linked collagenous peptides increased, and the thermal stabilization effect corresponded to an elongation by two units of the Gly-Pro-Hyp triad. The van't Hoff enthalpy (ΔH°) and the entropy (ΔS°) values of the cross-linked peptides increased with chain elongation, although the increments were smaller than those of the linear peptides. When the same chain lengths were compared, the ΔH° was increased and the ΔS° was nearly the same or increased by the cross-linking. These results suggest that the cross-linking of the collagenous peptides with the Lys-Lys-dimer template(s) for stabilization contributes to the enthalpic effect, rather than the entropic effect.     

ELECTRON PARAMAGNETIC RESONANCE STUDIES ON SPIN-LABELLING OF PEPSIN: EFFECTS OF TEMPERATURE,pH AND UREA ON ITS CONFORMATION

Pepsin was spin-labelled with N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidyl)bromo-acetamide, possibly at the active site, at a β-carboxyl group of a reactive aspartic acid. The spectrum of the spin-labelled pepsin showed that the spin probe was strongly immobilized (correlation time ≥10^-a sec). Spin-labelled pepsin was thermally denatured at various temperatures and electron paramagnetic resonance (e.p.r.) spectra were taken at various times. Rates of denaturation estimated from the e.p.r. spectra at various temperatures showed that the enthalpy and entropy of thermal denaturation of spin-labelled pepsin at pH3.5 were 48.0 ± 4.9 kcal/mole and 214.7 ± 14.5 e.u. respectively. Addition of conc. NaOH or 1 M acetate buffer at pH 6.0 sharpened e.p.r. spectra of the spin-labelled pepsin, indicating that the spin probe became mobilized by alkaline denaturation. Addition of urea caused unfolding of the protein which increased with the urea concentration, although only slight transition of conformational changes was observed in the e.p.r. spectra.     

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

Heat-denaturation of tryptophan synthase α-subunit from E. coli and two mutant proteins (Glu 49 ± 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°, C_p²⁰, has been found to be (0.43 ± 0.02) cal ± K^-1 ± g^-1, the unfolding heat capacity change, Δ_dC_p, (0.10 ± 0.01) cal ± K^-1 ± g^-1, and the unfolding enthalpy value extrapolated to 110°, Δ_dh¹¹⁰, (9.3 ± 0.5) cal ± g^-1 for the three proteins. The value of C_p²⁰ was larger than those found for fully compact protein and that of Δ_dh¹¹⁰ was smaller. Unfolding Gibbs energy, Δ_dG at 25° 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, Δ_dH, of the three proteins seemed to be the same and equal to (23.2 ± 1.2) kcal ± mol^-1 at 25°. As a consequence of the same value of Δ_dH and the different value in Δ_dG, substantial differences in unfolding entropy, Δ_dS, were found for the three proteins. The values of Δ_dG for the three proteins at 25° coincided with those from equilibrium methods of denaturation by guanidine hydrochloride.     

The effect of PEGylation on the stability of small therapeutic proteins

The influence of PEGylation on the thermal stability of small therapeutic proteins was evaluated using two model proteins. Changes in the midpoint of thermal unfolding and the ability to properly refold after thermal denaturation were monitored by differential scanning calorimetry (DSC) as a function of PEGylation and pH. The results showed that PEGylation increased the thermal stability of both model proteins as well as their ability to refold properly after thermal denaturation. The DSC results were compared to traditional accelerated stability data that were collected using size exclusion high performance liquid chromatography (SE-HPLC). The DSC data agreed reasonably well with those from SE-HPLC indicating that microcalorimetry can be an efficient screening tool for PEGylated proteins.     

The Effect of Temperature and pH on the Solubility of Quinolone Compounds: Estimation of Heat of Fusion

Although many reports involving fluoroquinolone agents have been published in the past decades, only a few address preformulation studies. In this paper, we describe the effect of temperature and pH on the aqueous solubility of two typically used quinolones, ciprofloxacin and norfloxacin. We measured the aqueous solubilities over the pH range of 5.5 to 9.5 at temperature of 6, 25, 30, and 40°C. The intrinsic solubilities and the thermodynamic dissociation constants were determined from solubility data and the temperature dependence of the intrinsic solubility was evaluated using vant Hoff and Hildebrand plots. The heat effusion was determined from these two plots. When the heat effusion was compared to that measured from differential scanning calorimetry (DSC) studies, we found that the Hildebrand method overestimated, and the vant Hoff equation underestimated, the heat of fusion. From the absolute values of the relative errors, the Hildebrand plot produced the better results. DSC results show that the heat effusion is 15.41 kcal/mol for ciprofloxacin and 7.88 kcal/mol for norfloxacin.     

The binding of food dyes with human serum albumin

The binding interactions between human serum albumin (HSA) and the edible food dyes amaranth, tartrazine and sunset yellow have been studied. Intrinsic association constants and the free energy changes associated with dye-protein binding at physiological pH for amaranth and tartrazine, and at two different pH values for sunset yellow have been calculated from ultrafiltration data. The temperature dependence (20–40°C) of the intrinsic association constants at pH 7.4 for amaranth-HSA and tartrazine-HSA mixtures have been measured, from which a plot of the van't Hoff isochore exhibits a marked change in slope around 30°C indicating a possible change in protein conformation. The number of dye binding sites on HSA is reported for all the above conditions. HSA-ligand binding enthalpies have been used in conjunction with the N-B transitional binding enthalpy for HSA, to calculate the enthalpy for the N-B transition when ligands are bound with the protein.     

Interdomain Interactions in the Chimeric Protein Toxin sCD4(178)-PE40: A Differential Scanning Calorimetry (DSC) Study

The thermal denaturation of the chimeric protein toxin known as sCD4(178)-PE40 (sCD4-PE40) was studied using differential scanning calorimetry (DSC). sCD4-PE40 consists of HIV-binding domains of the T-cell membrane protein known as CD4 and the cytotoxic domains of Pseudomonas exotoxin A (PE40). sCD4-PE40 undergoes two DSC transitions. An endothermic transition associated with unfolding of the CD4 and PE40 components occurs at approximately 46°C in buffered saline at pH 6.5. An exothermic transition associated with precipitation of unfolded protein occurs at higher temperatures. Both transitions are irreversible. DSC studies of solutions of pH 5.0 to 9.5 indicate that sCD4-PE40 shows maximal thermal stability at around pH 6.5. Variable pH experiments are also presented on solutions of sCD4(183) and PE40 revealing how these components denature as independent structural entities. sCD4(183) denaturation occurs at significantly higher temperatures than does the CD4 component of sCD4-PE40. PE40 denaturation occurs at the same temperatures as sCD4-PE40. These results suggest that the native CD4 and PE40 components are independent and non-interacting entities in the chimeric sCD4-PE40 molecule and that unfolding of the less-stable PE40 component induces unfolding of the CD4 component. These destabilizing interdomain interactions of sCD4-PE40 are in contrast to the stabilizing interactions which apparently exist in wild-type Pseudomonas exotoxin A between its PE40 domains and the cell binding domain of the native toxin (analogous to the CD4 component in sCD4-PE40). Reasons are discussed why the type of interdomain interactions observed for sCD4-PE40 might be the norm for chimeric proteins.Brian E. Collins: B.E.C.'s contribution was made while he was a summer intern at The Upjohn Company.     

Effects of Additives on Heat Denaturation of rhDNase in Solutions

Purpose. To study the thermal stability of recombinant human deoxyribonuclease I (rhDNase) in aqueous solutions. Methods. Differential scanning calorimetry (DSC) was used to measure the denaturation or melting temperature (T_m) and enthalpy (H_m) of rhDNase. The effects of denaturants (guanidine HC1 and urea) and additives (mainly divalent cations and disaccharides) were investigated at pH 6–7. Results. The T_m and H_m of rhDNase in pure water were measured as 67.4 °C and 18.0 J/g respectively, values typical of globular proteins. The melting peak disappeared on re-running the sample after cooling to room temperature, indicating that the thermal denaturation was irreversible. The latter was due to the occurrence of aggregation accompanying the unfolding process of rhDNase. Size exclusion chromatography indicated that during heat denaturation, rhDNase formed soluble high molecular weight aggregates with a molecular size >300kD estimated by the void volume. Of particular interest are the divalent cations: Ca²⁺ stabilizes rhDNase against thermal denaturation and elevates T_m and H_m while Mg²⁺, Mn²⁺ and Zn²⁺ destabilize it. Sugars also stabilize rhDNase. As expected, denaturants destabilize the protein and lower the T_m and H_m. All destabilization of rhDNase can be prevented by adding Ca²⁺ to the solutions. Conclusions. CaCl₂ and sugars were found to stabilize rhDNase against thermal denaturation while divalent cations, urea and guanidine HC1 destabilize the protein. The effects could be explained by a mixture of mechanisms. For Ca²⁺ the protective effect is believed to be due to an ordering of the rhDNase structure in its native state, and by prevention of breaking of a disulfide bridge, thus making it less susceptible to unfold under thermal stress.     

Simultaneous differential scanning calorimetry,X-ray diffraction and FTIR spectrometry in studies of ovalbumin denaturation

The new application of differential scanning calorimetry (DSC) and the susceptibility of ovalbumin to α-chymotrypsin gave a quantitative estimation of protein denaturation in solid ovalbumin. Solid ovalbumin in granules with 11% of water was heated at 100 °C in closed and nonclosed ampules. In order to compare effects of size and crystal structure, two proteins (bovine albumin and γ-globulin) were examined at similar conditions for the extent of denaturation. Ovalbumin and bovine albumin showed similar extents of denaturation, but γ-globulin, with a very different molecular mass, showed the maximal conformational changes. The enthalpy of denaturation was measured to elucidate the conformational changes in solid proteins. Its value was used for calculation of the degree of denaturation. The thermodynamic data associated with transition were calculated and the number of bonds broken during denaturation was determined. Intrinsic fluorescence was utilized in order to compare these two methods. Moreover, X-ray diffraction and FTIR spectrometry were applied to native and denatured proteins.     

Stability of subtilisins and related proteinases (subtilases)

The stability towards thermal and chemical (guanidine hydrochloride, GnHCl) denaturation of six inhibited subtilases (mesentericopeptidase, subtilisins BPN′, Carlsberg and DY, proteinase K and thermitase) has been investigated by kinetic and equilibrium studies. The unfolding processes were monitored by circular dichroic and fluorescence spectroscopy. Experiments in the absence and presence of extraneous calcium in the concentration range 2×10^?3-10^?1 M were performed. The presence of calcium in the weak calcium binding site changes the denaturation drastically. The heat- (or GnHCl-) induced unfolding curves obtained using CD spectroscopy show two independent transitions which seem not to have been resolved before. The presence of Ca²⁺ in the second (third in the case of thermitase) binding site increases the T_m, values by 11-21 °C and the δG_D(H₂O) values obtained from denaturation experiments in GnHCl by 6.7-7.2 kcal/mol when an extraneous Ca²⁺ concentration of 2 × 10^?2 M was used. One interpretation is that the initial step of denaturation in the presence of added calcium is the formation of a partially unfolded intermediate form, retaining a highly ordered structure with 60-85% of the a-helix structure of the native enzyme. This intermediate then unfolds at a temperature considerably higher than that of the same proteinases in the absence of added Ca²⁺. The free energy of stabilization of the intermediates is increased by 1.8-2.8 times in comparison with that for the unfolding reactions of the subtilases with empty Ca2/Ca3 binding sites. A second interpretation is that the two steps in the unfolding curves correspond to enzyme without and with calcium in the weak binding site. Fluorescence experiments confirm the mechanism involving the formation of intermediate states. The results are discussed in relation to the X-ray models of the six subtilases.     

Granulocyte-colony stimulating factor maintains a thermally stable,compact, partially folded structure at pH 2

At acidic pH many proteins exist in a partially unfolded form, called the “A” state. This is defined as a flexible, expanded structure with well-defined, usually native-like secondary structure, but no unique tertiary structure, and showing no cooperativity during thermal-induced denaturation. Granulocyte-colony stimulating factor (G-CSF), a four-helix bundle cytokine, maintains both thermal stability and tertiary structure at pH 2.O. We therefore examined the conformation and thermal unfolding of G-CSF at pH 2.0, 4.0 and 7.0 using circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR). The secondary structure of the molecule remains highly helical as the pH is lowered from 7.0 to 2.0. The tertiary structure of the protein is slightly different at each pH value, but even at pH 2.0 G-CSF maintains a regular three-dimensional structure. The structure is hydrodynamically compact at these different pH values, with no increase in Stake's radius even at pH 2.0.The thermal-induced denaturation of G-CSF was determined by monitoring changes in the CD or FTIR spectra. At pH 2.0 the temperature at which thermal-induced denaturation begins is higher than it is at pH 4.0 or 7.0, the thermal unfolding transition remains cooperative and some α-helical structure persists even at 86°C. At pH 4.0 and 7.0, secondary and tertiary structures disappear simultaneously during thermal denaturation, whereas at pH 2.0 small changes in the far-UV CD region begin to occur first, followed by the simultaneous cooperative loss of tertiary structure and much of the remaining secondary structure. The structure of G-CSF at pH 2.0 is thus revealed as compact, with a unique, three-dimensional structure, highly helical secondary structure, and most importantly, a cooperative thermal unfolding transition. G-CSF at acid pH thus does not adopt the “A” state.     

Study of protein conformational stability and integrity using calorimetry and FT-Raman spectroscopy correlated with enzymatic activity.

Maintaining protein conformational stability and integrity during formulation is critical for developing protein pharmaceuticals. Accordingly, high sensitivity differential scanning calorimetry (HSDSC) and Fourier transform (FT)-Raman spectroscopy were employed to assess conformational stabilities (thermal stability and folding reversibility) and structural integrities, respectively, for three model proteins: lysozyme, deoxyribonuclease I (DNase I) and lactate dehydrogenase (LDH) in lyophilised (as received) and spray-dried forms. Enzymatic assay after cooling of thermally denatured protein solutions from HSDSC determined if thermal transition reversibility was related to biological activity. HSDSC data showed that molecules from lyophilised lysozyme were able to refold better than the spray-dried form. This was confirmed by enzymatic assay. Moreover, enzymatic assay results revealed that lysozyme folding reversibility was related to the native structure of the protein that is essential for the biological activity. Thermal denaturation of DNase I and LDH samples in HSDSC was not reversible upon cooling of thermally denatured proteins (in contrast to lysozyme). Hence, it was decided to identify the effect of protein initial structures on its propensity to thermal denaturation via FT-Raman spectroscopy. In other words, proteins may denature with structural alterations due to stresses such as heat and the protein loses its enzymatic activity. Consequently, FT-Raman investigated the effects of spray drying and heating of solid DNase I and LDH samples, from differential scanning calorimetry, on protein conformational integrities. Lyophilised and spray-dried DNase I and LDH solid samples were heated to two temperatures, one before the apparent denaturation temperatures (Tm) and the other after the Tm. Samples heated below their Tm showed some alterations of the secondary structure and some enzymatic activity. HSDSC and FT-Raman spectroscopy are useful techniques to study protein conformations and their results correlate with those of enzymatic activity.     

Aristololactam-beta-D-glucoside. A new DNA binding monofunctional intercalating alkaloid

The binding of aristololactam-beta-D-glucoside to DNA is characterized by hypochromism and bathochromism in the absorption band, quenching of the fluorescence intensity, increase in the positive and negative ellipticity of DNA, enhancement of thermal transition temperature, sign and magnitude of thermodynamic parameters, increase of the contour length of sonicated rod-like DNA and induction of the unwinding-rewinding process of covalently closed superhelical DNA. Binding parameters determined from absorbance and fluorescence titration by Scatchard analysis, according to an excluded-site model, indicate a very high affinity towards DNA. The binding of the alkaloid is an exothermic process with Gibbs free energy of -7.4 kcal/mol, van't Hoff enthalpy of -13.8 kcal/mol and entropy of -21.5 cal/degree/mol at 25 degrees. On the basis of these observations it is concluded that aristololactam-beta-D-glucoside binds to DNA by a mechanism of intercalation.     

Effect of alkylation with different sized substituents on the conformation of ovomucoid,lysozyme and ovotransferrin

Conformational changes induced in ovomucoid, lysozyme and ovotransferrin on reductive addition of different sized substituents have been studied employing differential scanning calorimetry (DSC) and circular dichroic spectroscopy (CD). The thermograms obtained by DSC revealed that extensive introduction of methyl, isopropyl, cyclopentyl, cyclohexyl, benzyl or n-butyl groups has a detrimental effect on thermal stability (enthalpy of denaturation); the effect generally increases with the size of the substituent. Circular dichroic spectra were affected only to a very limited extent by the modifications, near-u.v. spectra remaining much the same while far-u.v. spectra displayed minor changes. The general conclusion drawn is that the modifications had only limited effects on the conformation of the proteins while, nonetheless, perturbing (or breaking) long-range intramolecular interactions so as to destabilize the structure. Derivatization of lysozyme and ovotransferrin with some of the larger groups has been reported to result in spontaneous precipitation of the proteins [Fretheim, K., Iwai, S. & Feeney, R.E. (1979) Int. J. Peptide Protein Res. 14, 451–456]. The present investigation indicates that precipitation was caused by (partial) denaturation (and ensuing aggregation) as a consequence of modification.     

Stability of DNA upon interaction with dimethyltin dichloride

Chemical and thermal denaturation of calf thymus DNA (as a multidomain macromolecule) have been investigated in the presence of high concentrations of dimethyltin dichloride (Me₂SnCl₂) over the temperature range (55–95°C) in 50.0 mM phosphate buffer at pH 7.6 using temperature scanning spectroscopy and calorimetry methods. Results showed that over the concentration range of 6–16 mM, Me₂SnCl₂ is a chemical denaturant and denatures the double-strand DNA in a three-state manner. The denaturation data are analyzed based on the effective Gibbs free energy (ΔG°_eff) approach and the chemical denaturation parameters including ΔG°_eff, m value and equilibrium unfolding constant (K _U) were obtained. Ultraviolet (UV) melting curves of the DNA at 260 nm as well as the calorimetric measurements were used to estimate the binding constants (K), melting enthalpy (ΔH°_m) and binding enthalpy (ΔH°_b). Furthermore, at low concentrations (up to 5 mM), Me₂SnCl₂ binds to the phosphate groups of DNA in an exothermic step and had no significant effect on double-strand DNA stability, confirmed by the fact that the T _m value did not change. However, high (denaturing) concentrations of Me₂SnCl₂ (more than 9 mM) caused considerable destabilization of DNA associated with the formation of a partially unfolded intermediate at 13.6 mM of Me₂SnCl₂. The formed intermediate showed a lower thermal transition temperature (T _m) by a magnitude of 10°C in relation to the native DNA. Finally, a new correlation is introduced for interpretation of thermal denaturation behavior of calf thymus DNA over the whole range of ligand (Me₂SnCl₂) concentration (0–16 mM).     

Solid state chemistry of proteins IV. what is the meaning of thermal denaturation in freeze dried proteins?

This research addresses the thermodynamic significance of the denaturation endotherm observed during differential scanning calorimetry (DSC) scans of proteins in dry formulations, such as freeze dried solids. Human growth hormone formulations are the chosen representative examples. We employ observations of denaturation temperature, glass transition temperature, and the differences between estimated molecular mobilities to argue that unfolding is under partial thermodynamic control. Further, unfolding during a DSC scan is simulated using a three state kinetic model, which is a two state unfolding model followed by aggregation. Kramers-type rate constants are used, where the preexponential term is dominated by viscous forces. Simulation results are in qualitative agreement with experiment, and clearly show that while the denaturation endotherm is impacted by irreversibility, caused by nonzero scan rate and aggregation, the position of the endotherm peak is changed only slightly. Thus, the denaturation peak is a good approximation for the thermodynamic denaturation temperature. Using data for denaturation temperature, heat of denaturation, and heat capacity of denaturation, free energy versus temperature curves were calculated. We find that even formulations with added saccharides are thermodynamically unstable near ambient temperature; significant denaturation in the solid state is prevented by low mobility.