Heat denaturation of human orosomucoid in water/methanol mixtures

Heat denaturation of orosomucoid in solutions of methanol concentrations ranging from 0 to 70% (v/v) has been studied by using circular dichroism, intrinsic protein fluorescence and thermal difference absorption spectroscopy. Regardless of its high saccharide content (40%), the highly cooperative denaturation transition of orosomucoid is fully reversible in neutral water solution. A two-state model has been successfully applied; the numerical analysis results in thermodynamical parameter values that are in close agreement with previously reported experimental data from calorimetric measurements. However, in solutions containing even minute concentrations of methanol (5%) the heat denaturation is irreversible. After cooling of the denatured protein the refolded molecules exhibit a higher α-helical content than the native one. Possibilities of methanol interaction with native and denatured protein molecule are discussed.     

Methanol-stabilized intermediates in the thermal unfolding of ribonuclease A: Characterization by 1H nuclear magnetic resonance

The thermal unfolding of ribonuclease A has been studied in solutions of 25, 35 and 50% methanol ($\text{v}\text{v}$), using 360 MHz proton magnetic resonance spectroscopy. Several observations indicate that the native structure of the protein in methanol cryosolvents is very similar to that in aqueous solution. A detailed analysis of the unfolding process has been made using the C-2 protons of the imidazole side-chains of the four histidine residues. As denaturation proceeds new resonances appear, whose chemical shifts correspond to neither native nor fully unfolded species. These have been assigned to particular His residues by selective deuteration studies. The thermal denaturation transitions reveal a multiphasic process in each of the solvents, and become less co-operative with increasing concentrations of methanol. The denaturation is fully reversible with no evidence of hysteresis.The new resonances that appear during the unfolding process are attributed to partially folded species, which are stabilized by the presence of the relatively hydrophobic methanol. Based on the temperature dependence of the chemical shifts and the relative areas of the various resonances, a detailed sequence of events has been proposed to describe the unfolding process. Key features include the initial general loosening of the two domains, the subsequent movement of the upper S-peptide region (residues 13 to 25) away from the main body of the protein, followed by partial separation of the sheet structure and full exposure of the N-terminal helix, leading to complete separation of the “winged domains”, and ultimately the loss of the residual sheet and helix structure.     

Conformational stability of HPr: the histidine-containing phosphocarrier protein from Bacillus subtilis.

The conformational stability of the histidine-containing phosphocarrier protein (HPr) from Bacillus subtilis has been determined using a combination of thermal unfolding and solvent denaturation experiments. The urea-induced denaturation of HPr was monitored spectroscopically at fixed temperatures and thermal unfolding was performed in the presence of fixed concentrations of urea. These data were analyzed in several different ways to afford a measure of the cardinal parameters (delta Hg, Tg, delta Sg, and delta Cp) that describe the thermodynamics of folding for HPr. The method of Pace and Laurents (Pace CN, Laurents DV, 1989, Biochemistry 28:2520-2525) was used to estimate delta Cp as was a global analysis of the thermal- and urea-induced unfolding data. Each method used to analyze the data gives a similar value for delta Cp (1,170 +/- 50 cal mol-1K-1). Despite the high melting temperature for HPr (Tg = 73.5 degrees C), the maximum stability of the protein, which occurs at 26 degrees C, is quite modest (delta Gs = 4.2 kcal mol-1). In the presence of moderate concentrations of urea, HPr exhibits cold denaturation, and thus a complete stability curve for HPr, including a measure of delta Cp, can be achieved using the method of Chen and Schellman (Chen B, Schellman JA, 1989, Biochemistry 28:685-691). A comparison of the different methods for the analysis of solvent denaturation curves is provided and the effects of urea on the thermal stability of this small globular protein are discussed. The methods presented will be of general utility in the characterization of the stability curve for many small proteins.     

Temperature- and concentration-dependence of compatibility of the organic osmolyte beta-dimethylsulfoniopropionate.

The effects of the organic osmolyte beta-dimethylsulfoniopropionate (DMSP) on the structural stability of three model proteins were examined to determine whether DMSP, like the structurally similar solute dimethyl sulfoxide (DMSO), is compatible with native protein structure at low, but not elevated, temperatures. DMSP stabilized phosphofructokinase under conditions of cold-induced denaturation. Thus, DMSP, like DMSO, may be an effective protein cryoprotectant. However, DMSP was not an effective stabilizer of protein structure under conditions of heat denaturation. Whereas low (0.2 M) concentrations of DMSP stabilized lactate dehydrogenase against inactivation at 50 degrees C, higher DMSP concentrations were ineffective. DMSP favored the denaturation of glutamate dehydrogenase at all DMSP concentrations tested. DMSP may be a compatible osmotic solute only under conditions of moderate temperature and low, yet physiological, concentrations. The mechanistic basis of DMSP's temperature- and concentration-dependent effects and the possible roles played by adaptation temperature and severity of osmotic stress in the evolutionary selection of organic osmolytes are discussed.     

The denaturation of ribonuclease A by combinations of urea and salt denaturants.

When urea is added to ribonuclease A that has already been denatured by salt (CaCl₂, LiClO₄ or LiCl were used), a second co-operative transition occurs, supporting the previous demonstration that these salts cause only partial denaturation. Also we have studied the effect of the salts on the urea denaturation, and the effect of urea on the salt denaturation. At low concentrations urea makes the salt transitions occur at lower concentrations, but at higher concentrations it changes the transition so that the completely disordered protein found in urea is produced by the salt. At low concentrations the salts actually stabilize the protein against denaturation by urea, but at higher concentrations they destabilize it. The data are presented in “phase diagrams” which are found to be very useful for such three-component systems.     

Characterization of the unfolding of ribonuclease A in aqueous methanol solvents

The effect of methanol on the thermal denaturation of ribonuclease A has been investigated over the -40 to 70 degrees C range. The transition was fully reversible to at least 60% (v/v) methanol at an apparent pH of cryosolvent (pH) of 3.0 and was examined at methanol concentrations as high as 80%. The unfolding transition, as monitored by absorbance change at 286 nm, became progressively broader and occurred at increasingly lower temperatures as the alcohol concentration increased. In 50% methanol, increasing the pH from 2 to 6 shifted the transition to higher temperature. A substantial decrease in cooperativity was noted at the more acidic conditions. On the other hand, increasing concentrations of guanidine hydrochloride in 50% methanol caused the transition to shift to lower temperatures with little effect on the cooperativity. The observed effects on the cooperativity of the unfolding transition suggest that methanol and lower temperatures may increase the concentration of partially folded intermediate states in the unfolding of ribonuclease. Comparison of the transition in 50% methanol as determined by absorbance or fluorescence, which monitor the degree of exposure of buried tyrosines and hence the tertiary structure, to that determined by far-UV circular dichroism, which monitors secondary structure, indicated that the major unfolding transition occurred at a higher temperature in the latter case. Thus, the tertiary structure is lost at a lower temperature than the secondary structure. This observation is consistent with a model of protein folding in which initially formed regions of secondary structure pack together, predominantly by hydrophobic interactions, to give the tertiary structure.(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.     

Interaction of alcohols with proteins

The kinetics of denaturation of egg albumin have been determined for methanol, ethanol, propanol, and butanol. The reactions are first order in respect to protein but between 11th and 18th order for the alcohols. The denaturation reaction is characterized by a large temperature coefficient with little or no dependence on pH. There is a marked change of pH when proteins are denatured. A series of eight proteins has been studied. There is surprisingly little difference in susceptibility to alcohol denaturation between the various proteins. Methanol, ethanol, propanol, and butanol are strongly bound to egg albumin—butanol being the most strongly bound. The binding of alcohol is probably accompanied by protein dehydration. The polyhydric alcohols' behavior is much different. These alcohols do not denature proteins and the protein is hydrated. Sucrose produces the greatest degree of hydration.     

Kinetics and thermodynamics of thermal denaturation in acyl carrier protein.

The denaturation of Escherichia coli acyl carrier protein (ACP) in buffers containing both monovalent and divalent cations was followed by variable-temperature NMR and differential scanning calorimetry. Both high concentrations of monovalent salts (Na+) and moderate concentrations of divalent salts (Ca2+) raise the denaturation temperature, but calorimetry indicates that a significant increase in the enthalpy of denaturation is obtained only with the addition of a divalent salt. NMR experiments in both low ionic strength monovalent buffers and low ionic strength monovalent buffers containing calcium ions show exchange between native and denatured forms to be slow on the NMR time scale. However, in high ionic strength monovalent buffers, where the temperature of denaturation is elevated as it is in the presence of Ca2+, the transition is fast on the NMR time scale. These results suggest that monovalent and divalent cations may act to stabilize ACP in different ways. Monovalent ions may nonspecifically balance the intrinsic negative charge of this protein in a way that is similar for native, denatured, and intermediate forms. Divalent cations provide stability by binding to specific sites present only in the native state.     

Albumin denaturation during ultrafiltration: effects of operating conditions and consequences on membrane fouling

Ultrafiltration of high-purity grade bovine serum albumin has been carried out under various temperature between 5 and 30 degrees C and at various cross-flow velocities, pressures, and concentrations with the aim of studying protein denaturation and its consequences on the process. Three different pump heads have been tested. Denaturation of proteins in solution has been monitored by laser light scattering and size exclusion chromatography. The rate of protein denaturation increases with temperature, cross-flow, and time. It is observed that membrane fouling is different whether denaturation has occurred or not. Under high-concentration polarization, denaturation can occur in the boundary layer if the wall concentration exceeds 400 g/L. It is shown how the residence time, operating temperature, and pressure play an important part in membrane fouling. This can provide guidelines for process design and control.     

The reaction of oxyhemoglobin with 1,1-diphenyl-2-picrylhydrazine and 2,2-diphenyl-1-picrylhydrazyl

In an effort to elucidate the mechanism of the initiation reaction of the denaturation of oxyhemoglobin, I, to methemoglobin, II, by hydrazines, we have investigated by electron paramagnetic resonance, EPR, and visible spectrophotometry at 22 degrees C, pH 7.4, the reaction of I with 1,1-diphenyl-2-picrylhydrazine, III, and 2,2-diphenyl-1-picrylhydrazyl, IV, in dimethylsulphoxide/buffer and methanol/buffer mixtures, these organic solvents included at a concentration of 10 v/v% to render III and IV soluble while not causing appreciable denaturation of I. In both buffer mixtures, the results obtained were the same. For the I/III reaction mixtures, although the spectrophotometric data obtained showed denaturation to occur, there was no EPR evidence for formation of IV, contrary to expectation based on the chemical structure of III. The EPR observations on each I/IV reaction mixture showed a rapid decrease in IV signal intensity to a value that, depending on the initial reactant concentrations, was either below the detection limit or, when measurable, constant with time. The results of similar EPR measurements on analogous II/IV reaction mixtures were the same. These EPR results are compatible with the idea that IV forms a complex with the protein moiety of I and II, and show that the I/III reaction could yield IV and thus involve a one-electron transfer process.     

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.     

Four-state equilibrium unfolding of an scFv antibody fragment

The conformational stability of a single-chain Fv antibody fragment against a hepatitis B surface antigen (anti-HBsAg scFv) has been studied by urea and temperature denaturation followed by fluorescence and circular dichroism. At neutral pH and low protein concentration, it is a well-folded monomer, and its urea and thermal denaturations are reversible. The noncoincidence of the fluorescence and circular dichroism transitions indicates the accumulation in the urea denaturation of an intermediate (I(1)) not previously described in scFv molecules. In addition, at higher urea concentrations, a red-shift in the fluorescence emission maximum reveals an additional intermediate (I(2)), already reported in the denaturation of other scFvs. The urea equilibrium unfolding of the anti-HBsAg scFv is thus four-state. A similar four-state behavior is observed in the thermal unfolding although the intermediates involved are not identical to those found in the urea denaturation. Global analysis of the thermal unfolding data suggests that the first intermediate displays substantial secondary structure and some well-defined tertiary interactions while the second one lacks well-defined tertiary interactions but is compact and unfolds at higher temperature in a noncooperative fashion. Global analysis of the urea unfolding data (together with the modeled structure of the scFv) provides insights into the conformation of the chemical denaturation intermediates and allows calculation of the N-I(1), I(1)-I(2), and I(2)-D free energy differences. Interestingly, although the N-D free energy difference is very large, the N-I(1) one, representing the "relevant" conformational stability of the scFv, is small.     

Nonideality and protein thermal denaturation

We studied the thermal denaturation of eglin c by using CD spectropolarimetry and differential scanning calorimetry (DSC). At low protein concentrations, denaturation is consistent with the classical two-state model. At concentrations greater than several hundred microM, however, the calorimetric enthalpy and the midpoint transition temperature increase with increasing protein concentration. These observations suggested the presence of intermediates and/or native state aggregation. However, the transitions are symmetric, suggesting that intermediates are absent, the DSC data do not fit models that include aggregation, and analytical ultracentrifugation (AUC) data show that native eglin c is monomeric. Instead, the AUC data show that eglin c solutions are nonideal. Analysis of the AUC data gives a second virial coefficient that is close to values calculated from theory and the DSC data are consistent with the behavior expected for nonideal solutions. We conclude that the concentration dependence is caused by differential nonideality of the native and denatured states. The nondeality arises from the high charge of the protein at acid pH and is exacerbated by low buffer concentrations. Our conclusion may explain differences between van't Hoff and calorimetric denaturation enthalpies observed for other proteins whose behavior is otherwise consistent with the classical two-state model.     

Denaturation of proteins in methanol/water mixtures

The solvophobic theory developed earlier by Sinanoglu introducing the use of molecular surface areas and microthermodynamic surface and interfacial tensions at molecular dimensions is applied to the interpretation of calorimetric data on denaturation of lysozyme in a wide range of methanol/water mixtures. The experimental values of standard unitary free energies of denaturation correlate well with our predictions. The molecular surface area change of the protein upon denaturation is evaluated using the solvophobic theory. The maximum in the stability of the native form of the protein is predicted to occur at 8% (v/v) methanol. This is found to be in agreement with the experimental results.     

The cold-induced denaturation of lactate dehydrogenase at sub-zero temperatures in the absence of perturbants

The cold-induced denaturation of lactate dehydrogenase has been determined in an unfrozen, cryoprotectant free solution at sub-zero temperatures. The cold-induced denaturation temperature (TL) has been found to be -28 degrees C. The results for the first time clearly establish that temperature alone can induce denaturation in a cooled protein solution. The validity of earlier data, obtained in the presence of perturbants (particularly pH or guanidinium chloride), is discussed.     

Thermal denaturation of human gamma-interferon. A calorimetric and spectroscopic study.

The thermal denaturation of a recombinant human gamma-interferon has been studied as a function of pH in the range from 2 to 10 and buffer concentration in the range from 5 to 100 mM by differential scanning calorimetry, circular dichroism, fluorescence, 1H NMR, and biological activity measurements. The thermal transitions are irreversible at high buffer concentrations at all pH values studied, although they are reversible between pH 3.5 and 5.4 at low buffer concentrations. The denaturation enthalpy, DeltaH(Tm), at denaturation temperature Tm was a function of both Tm and the buffer concentration, and this resulted in heat capacity changes decreasing with buffer concentration. When the denaturation enthalpies were corrected for Tm dependence, they did not appear to change versus pH. The denaturation entropies, however, appeared to decrease with pH, leading to a small but appreciable increase in the stability of the protein with pH. The difference between the number of moles of protons stoichiometrically bound to a mole of protein in the native and thermally denatured state, was calculated from the variation of Tm versus pH at each buffer concentration. The values obtained appear to depend on pH alone rather than upon temperature or buffer concentration, a result which agrees with the invariance of the denaturation enthalpies with pH. This dependence was fitted to the titration curve of a group with a pK of 5.4.     

Effect of organic solvents on the structure and activity of moderately halophilic Bacillus sp. EMB9 protease

Halophilic enzymes have been manifested for their stability and catalytic abilities under harsh operational conditions. These have been documented to withstand denaturation in presence of high temperature, pH, presence of organic solvents and chaotropic agents. The present study aims at understanding the stability and activity of a halophilic Bacillus sp. EMB9 protease in organic solvents. The protease was uniquely stable in polar solvents. A clear correlation was evident between the protease function and conformational transitions, validated by CD and fluorescence spectral studies. The study affirms that preservation of protein structure, possibly due to charge screening of the protein surface by Ca²⁺ and Na⁺ ions provides stability against organic solvents and averts denaturation. Salt was also found to exert a protective effect on dialyzed protease against chaotropism of solvents. Presence of 1 % (w/v) NaCl restored the activity in the dialyzed protease and prevented denaturation in methanol, toluene and n-decane. The work will have further implication on discerning protein folding in saline as well as non-aqueous environments.     

Fluorescence and circular dichroism spectroscopy of cytochrome c in alkylammonium formate ionic liquids

The structural stability of cytochrome c has been studied in alkylammonium formate (AAF) ionic liquids such as methylammonium formate (MAF) and ethylammonium formate (EAF) by fluorescence and circular dichroism (CD) spectroscopy. At room temperature, the native structure of cytochrome c is maintained in relatively high ionic liquid concentrations (50-70% AAF/water or AAF/phosphate buffer pH 7.0) in contrast with denaturation of cytochrome c in similar solutions of methanol or acetonitrile with water or buffer cosolvents. Fluorescence and CD spectra indicate that the conformation of cytochrome c is maintained in 20% AAF-80% water from 30 to 50 °C. No such temperature stability is found in 80% AAF-20% water. About one-third of the enzyme activity of cytochrome c in 80% AAF-20% water can be maintained as compared with phosphate buffer, and this is greater than the activities measured in corresponding methanol and acetonitrile aqueous solutions. This biophysical study shows that AAFs have potential application as organic solvent replacements at moderate temperature in the mobile phase for the separation of proteins in their native form by reversed phase liquid chromatography.