Morphologies and conformation transition of lentinan in aqueous NaOH solution

Molecular morphologies and conformation transition of lentinan, a beta-(1-->3)-D-glucan from Lentinus edodes, were studied in aqueous NaOH solution by atomic force microscopy (AFM), viscometry, multiangle laser light scattering, and optical rotation measurements. The results revealed that lentinan exists as triple-helical chains and as single random-coil chains at NaOH concentration lower than 0.05M and higher than 0.08M, respectively. Moreover, the dramatic changes in weight-average molecular weight Mw, radius of gyration [s2](1/2), intrinsic viscosity [eta], as well as specific optical rotation at 589 nm [alpha]589 occurred in a narrow range of NaOH concentration between 0.05 and 0.08M NaOH, indicating that the helix-coil conformation transition of lentinan was carried out more easily than that of native schizophyllan and scleroglucan, and was irreversible. For the first time, we confirmed that the denatured lentinan molecule, which was dissolved in 0.15M NaOH to be disrupted into single coil chains, could be renatured as triple helical chain by dialyzing against abundant water in the regenerated cellulose tube at ambient temperature (15 degrees C). In view of the AFM image, lentinan in aqueous solution exhibited the linear, circular, and branched species of triple helix compared with native linear schizophyllan or scleroglucan.     

Mechanism and dynamics of conformational ordering in xanthan polysaccharide

The thermally induced order-disorder transition of xanthan (extracellular bacterial polysaccharide from Xanthomonas campestris) has been investigated by optical rotation, differential scanning calorimetry, stopped-flow reaction kinetics and low-angle laser light scattering, and the results have been analysed in terms of Zimm -Bragg helix-coil transition theory. The reciprocal of the transition midpoint temperature (Tm) varies linearly with the logarithm of cation (K+) the salt dependence of Tm, is in agreement with Manning polyelectrolyte theory the ordered structure. The associated increase in cation binding, calculated from the salt dependence of tm, is in agreement with the Manning polyelectrolyte theory for one of the candidate structures from X-ray diffraction, a 5(1) single helix stabilized by packing of side-chains along the polymer backbone, but not for the alternative double-helix structure that has also been proposed. At each salt concentration, the two fundamental parameters of the Zimm -Bragg theory, s and sigma, were calculated. The equilibrium constant for growth of the ordered structure (s) is derived directly from calorimetric measurement of transition enthalpy (delta Hcal ), and sigma, which quantifies the relative instability of the helix nucleus, is derived from the ratio of delta Hcal to the apparent transition enthalpy (delta Happ ) obtained by van't Hoff analysis of the optical rotation data. The temperature course of conformational ordering calculated theoretically is in good quantitative agreement with experimental results from both optical rotation and scanning calorimetry. The calculated average length of stable, ordered chain-sequences increases with decreasing temperature, but equals or exceeds the total chain length from light scattering only at temperatures more than approximately equal to 70 K below Tm, suggesting that ordered and disordered regions may co-exist within the same xanthan molecule. Consistent with this interpretation, the observed rate of conformational ordering increases sharply under conditions where the starting solution for dynamic measurements is partially ordered, suggesting that ordered sequences within each chain may act as helix nuclei for adjacent disordered regions, so that helix growth, rather than the slower nucleation process, becomes rate limiting.     

A new bacterial polysaccharide (YAS34). I. Characterization of the conformations and conformational transition

This paper concerns the study of the conformational transition of a new exopolysaccharide (YAS34) using experimental techniques such as optical rotation, conductimetric and microcalorimetric measurements as a function of temperature. The behaviors of this polysaccharide in the acid or sodium salt form are compared; a deacetylated sample is also prepared to demonstrate the role of substituents. For the native structure (never heated), a conformational transition is observed but the deacetylated polysaccharide exhibits no ordered conformation. Multidetection size exclusion chromatography (SEC) analyses and conductimetric experiments allowed to determine the nature of each conformation and the molecular dimensions. From these results, it is suggested that the native conformation is a double helix which by heating over T(m) (temperature corresponding to half conformational transition) dissociates into disordered single chains. In the acid and sodium salt forms, by cooling below T(m), an ordered conformation is restored. This conformation seems to be an intramolecular double helix 'hairpin-like turn' (called renatured conformation). Nevertheless an irreversible denaturation is obtained progressively in the sodium salt form when the time of heating over T(m) increases. The conformation of the deacetylated polysaccharide corresponds to that of a single flexible chain (disordered conformation). The conformational transition for the native conformation was studied also in relation to the polyelectrolytic character of the polysaccharide: stability as a function of salt nature and salt and polymer concentrations was investigated for the polymer initially in the sodium and acid forms.     

Thermodynamic characterization of cytochrome c at low pH. Observation of the molten globule state and of the cold denaturation process.

Several reports have pointed out the existence of intermediate states (both kinetic and equilibrium intermediate) between the native and the denatured states. The molten globule state, a compact intermediate state in which the secondary structure is formed but the tertiary structure fluctuates considerably, is currently being studied intensively because of its possible implication in the folding process of several proteins. We have examined the thermal stability of horse cytochrome c at low pH between 2.0 and 3.2 and different potassium chloride concentrations by absorbance of the Soret band, far and near-ultraviolet circular dichroism (u.v. c.d.) and tryptophan fluorescence using a multidimensional spectrophotometer. The concentration of potassium chloride ranged from 0 M to 0.5 M. The experimental thermal denaturation curves show that: (1) the helical content of cytochrome c remains stable at higher temperature when the concentration of salt is increased; whereas (2) the extent of ordering of the tertiary structure is weakly dependent on salt concentration; and (3) for cytochrome c, the stabilization of the molten globule state is induced by the binding of anions. Other salts such as NaCl, LiCl, potassium ferricyanide (K3Fe(CN)6) and Na2SO4 may also be used to stabilize the molten globule state. The thermodynamic analysis of the denaturation curves of c.d. at 222 nm and c.d. at 282 nm shows that, whereas a two-state (native and denatured) transition is observed at low-salt concentration, the far and near-u.v. c.d. melting curves of cytochrome c do not coincide with each other at high-salt concentration, and a minimum of three different thermodynamic states (IIb, intermediate or IIc, and denatured) is necessary to achieve a sufficient analysis. The intermediate state (called IIc) is attributed to the molten globule state because of its high secondary structure content and the absence of tertiary structure. Therefore, at low pH, cytochrome c is present in at least four states (native, IIb, IIc and denatured) depending on the salt concentration and temperature. The thermodynamic parameters, i.e. the Gibbs free energy differences (delta G), the enthalpy differences (delta H), the midpoint temperatures (Tm) of the transition (IIb in equilibrium intermediate (IIc in equilibrium denatured) are determined. We also give estimates of the heat capacity differences (delta Cp) from the temperature dependence of the enthalpy differences. The enthalpy change and the heat capacity difference of the IIc in equilibrium denatured transition are non-zero. The number of charges (protons or chloride anions) released upon transitions are determined by analysing the pH and chloride anion concentration dependence of the Gibbs free energy.(ABSTRACT TRUNCATED AT 400 WORDS)     

Conformational and thermodynamic properties of apo A-1 of human plasma high density lipoproteins.

Conformational changes of apo A-1, the principal apoprotein of human plasma high density lipoprotein, have been studied by differential scanning calorimetry and ultraviolet difference spectroscopy as a function of temperature, pH, concentration of apoprotein, and urea concentration. Calorimetry shows that apo A-1 (5 to 40 mg/ml, pH 9.2) undergoes a two-state, reversible denaturation (enthalpy = 64 +/- 8.9 kcal/mole), between 43--71 degrees (midpoint temperature, Tm = 54 degrees), associated with a rise in heat capacity (deltaCvd) of 2.4 +/- 0.5 kcal/mole/degrees C. Apo A-1 (0.2 to 0.4 mg/ml, pH 9.2) develops a negative difference spectrum between 42--70 degrees, with Tm = 53 degrees. The enthalpy (deltaH = 59 +/- 5.7 kcal/mole at Tm) and heat capacity change (2.7 +/- 0.9 kcal/mole/degrees C) in the spectroscopic experiments were not significantly different from the calorimetric values. Below pH 9 and above pH 11, the calorimetric Tm and deltaH of denaturation are decreased. In the pH range of reversible denaturation (6.5 to 11.8), delatH and Tm are linearly related, showing that the heat capacity change (ddeltaH/dT) associated with denaturation is independent of Tm. In urea solutions, the calorimetric Tm and deltaH of denaturation are decreased. At 25 degrees, apo A-1 develops a negative difference spectrum between 1.4 and 3 M urea. Fifty per cent of the spectral change occurs in 2.4 M urea, which corresponds to the urea concentration obtained by extrapolation of the calorimetric Tm to 25 degrees. In urea solution of less than 0.75 M there is hyperchromicity at 285 nm (delta epsilon = 264 in 0.75 M urea), indicating strong interaction of aromatic amino acid residues in the native molecule with the solvent. Spectrophotometric titration of apo A-1 shows that 6.6 of the 7 tyrosine groups of apo A-1 titrate at pH less than 11.9, with similar titration curves obtained in aqueous solutions and in 6 M urea. The free energy of stabilization (deltaG) of the native conformation of apo A-1 was estimated, (a) at 37 degrees, using the calorimetric deltaA and deltaCvd, and (b) at 25 degrees, by extrapolation of spectroscopic data to zero urea concentration. The values (deltaG (37 degrees) = 2.4 and deltaG (25 degrees) = 2.7 kcal/mole) are small compared to typical globular proteins, indicating that native apo A-1 has a loosely folded tertiary structure. The low values of deltaG reflect the high degree of exposure of hydrophobic areas in the native protein molecule. The loosely folded conformation of apo A-1 allows extensive binding of lipid, since this can involve both surface hydrophobic sites and hydrophobic areas exposed by a cooperative, low energy unfolding process.     

Conformation and structure of mildly heat-treated ovalbumin in dilute solutions and gel formation at higher protein concentrations

The conformation of heat-denatured ovalbumin aggregates has been examined at several concentrations and pH values, using measurements of optical rotation dispersion (ORD), circular dichroism (CD) and viscosity. The protein was subjected to heating at relatively low temperatures, ranging from 48.5 to 76 degrees; the particular temperature chosen depended on pH. The heat treatment was sufficient to remove the ability of the molecules to absorb heat on re-heating. The denatured molecules were shown to be rather compact, i.e. not much larger than the native molecule, and to retain a significant amount of secondary structure; this was also the case for molecules present in small aggregates. It is suggested that this type of ovalbumin monomer builds three-dimensional networks in denatured solutions at higher concentrations, and that gelation should be looked upon as arising from surface contacts between hydrated globules. The present results also imply that such globules have gelation properties which depend on whether pH is acidic or basic compared to the isoelectric point of the protein.     

Water and urea interactions with the native and unfolded forms of a beta-barrel protein

A fundamental understanding of protein stability and the mechanism of denaturant action must ultimately rest on detailed knowledge about the structure, solvation, and energetics of the denatured state. Here, we use (17)O and (2)H magnetic relaxation dispersion (MRD) to study urea-induced denaturation of intestinal fatty acid-binding protein (I-FABP). MRD is among the few methods that can provide molecular-level information about protein solvation in native as well as denatured states, and it is used here to simultaneously monitor the interactions of urea and water with the unfolding protein. Whereas CD shows an apparently two-state transition, MRD reveals a more complex process involving at least two intermediates. At least one water molecule binds persistently (with residence time >10 nsec) to the protein even in 7.5 M urea, where the large internal binding cavity is disrupted and CD indicates a fully denatured protein. This may be the water molecule buried near the small hydrophobic folding core at the D-E turn in the native protein. The MRD data also provide insights about transient (residence time <1 nsec) interactions of urea and water with the native and denatured protein. In the denatured state, both water and urea rotation is much more retarded than for a fully solvated polypeptide. The MRD results support a picture of the denatured state where solvent penetrates relatively compact clusters of polypeptide segments.     

Influence of the pyruvate content of xanthan on macromolecular association in solution

The unusual increase in viscosity and pseudoplasticity often observed when salts are added to moderately concentrated aqueous solutions of xanthan gum is shown to arise from an increase in the extent of macromolecular association. The fractional change in viscosity on addition of KCl to salt-free 1% (w/v) solutions of purified polysaccharide in the K⁺ salt form is found to be positive only when the degree of pyruvate substitution (fraction of side chains which carry pyruvate ketal substituents) exceeds ≈0.31. Above this value, the fractional change in viscosity increases with further increase in the degree of pyruvate substitution. These differences cannot arise from different degrees of conformational ordering, since the magnitude of the thermally induced order disorder transition (monitored by optical rotation at low ionic strength) is independent of pyruvate content. The temperature at the transition midpoint, however, falls with increasing degree of pyruvate substitution. This is attributed to destabilization of the ordered structure by intramolecular electrostatic repulsion between pyruvate groups, and stabilization through apolar interactions of acetate methyl groups. Viscosity-concentration relationships show changes of slope which mark the onset of macromolecular association. Association commences at lower concentrations when ionic strength and degree of pyruvate substitution are high. It is suggested that once electrostatic repulsions have been diminished at high ionic strength, association is promoted by intermolecular apolar interactions of pyruvate methyl groups, which are suitably near the periphery of the helical conformation.     

Localization of the structural change induced in tRNA fMET (Escherichia coli) by acidic pH.

We have compared the molecular mechanism of thermal unfolding for native tRNA fMet (Escherichia coli) and the denatured species produced by annealing at pH 4.3. Relaxation kinetic measurements reveal that the transitions assigned to melting of TphiC, anticodon, and acceptor stem helices at neutral pH remain essentially unaltered at pH 4.3, but the transition corresponding to coupled melting of tertiary structure and dihydrouridine helix is greatly affected. The Tm of this region is more than 20 degrees higher at pH 4.3 and it has a larger enthalpy formation than in the native state. The transition dynamics are also considerably changed. In contrast to the native structure, tRNA fMet1 and tRNA fMet3 have similar tertiary structure stabilities at pH 4.3. We conclude that the structural difference between native and acid-denatured forms is localized in the tertiary structure-dihydrouridine helix cooperative interaction region of the molecule.     

Thermodynamic characterization of an equilibrium folding intermediate of staphylococcal nuclease.

High-sensitivity differential scanning calorimetry and CD spectroscopy have been used to probe the structural stability and measure the folding/unfolding thermodynamics of a Pro117-->Gly variant of staphylococcal nuclease. It is shown that at neutral pH the thermal denaturation of this protein is well accounted for by a 2-state mechanism and that the thermally denatured state is a fully hydrated unfolded polypeptide. At pH 3.5, thermal denaturation results in a compact denatured state in which most, if not all, of the helical structure is missing and the beta subdomain apparently remains largely intact. At pH 3.0, no thermal transition is observed and the molecule exists in the compact denatured state within the 0-100 degrees C temperature interval. At high salt concentration and pH 3.5, the thermal unfolding transition exhibits 2 cooperative peaks in the heat capacity function, the first one corresponding to the transition from the native to the intermediate state and the second one to the transition from the intermediate to the unfolded state. As is the case with other proteins, the enthalpy of the intermediate is higher than that of the unfolded state at low temperatures, indicating that, under those conditions, its stabilization must be of an entropic origin. The folding intermediate has been modeled by structural thermodynamic calculations. Structure-based thermodynamic calculations also predict that the most probable intermediate is one in which the beta subdomain is essentially intact and the rest of the molecule unfolded, in agreement with the experimental data. The structural features of the equilibrium intermediate are similar to those of a kinetic intermediate previously characterized by hydrogen exchange and NMR spectroscopy.     

Structure and properties of a bacterial polysaccharide from a Klebsiella strain (ATCC 12657)

The chemical structure and the rheological behavior of the Klebsiella polysaccharide ATCC 12657 was studied and compared with data described in the literature and obtained for similar polysaccharides. The acetylated polysaccharide presents in solution a normal viscoelastic behavior with no evidence of an ordered conformation whatever the experimental conditions are. The deacetylated form can induce the formation of physical gels, in the presence of salt excess or ethanol. Microcalorimetry, optical rotation, and rheology experiments demonstrate that a thermally reversible and highly cooperative conformational transition occurs at the same temperature than a sol-gel transition. The melting of the gel and the conformational transition temperatures are dependent on the nature of cations and ionic concentration, whereas the gel strength is only influenced by polymer concentration.     

Thermally induced conformation change of succinoglycan in aqueous sodium chloride

Static and dynamic light scattering, viscosity, and optical rotation measurements have been made at eight different temperatures between 25 and 75 degrees C on two succinoglycan samples (sodium salt) with weight-average molecular weights M(w) of 7.14 x 10(5) and 3.54 x 10(5) (at 25 degrees C) in 0.01 M aqueous NaCl to investigate the thermally induced order-disorder conformation change of the polysaccharide. Additionally, viscometry and polarimetry have been performed for a sodium salt sample (M(w) = 4.55 x 10(5) at 25 degrees C) whose M(w), z-average radius of gyration (z)(1/2), and hydrodynamic radius R(H) in the aqueous salt had been determined previously. As the temperature increases, M(w), (z)(1/2), R(H), and the intrinsic viscosity for every sample sharply decrease around 55 degrees C where the specific rotation at 300 nm sigmoidally increases. In particular, M(w) at 25 degrees C (i.e., in the ordered helical state) is twice as large as that at 75 degrees C (i.e., in the disordered state). These findings substantiate that the ordered structure is composed of two chains and hence is a double helix. Data analysis shows that this helix at 25 degrees C is characterized by an unperturbed wormlike chain with a helix pitch of about 2 nm (per repeating unit) and a persistence length of about 50 nm and that upon heating, it dissociates directly (i.e., in all-or-none fashion) to disordered chains of a similar contour length but with a much smaller persistence length of about 10 nm. The temperature dependence of the light scattering second viral coefficient is discussed in relation to the association of disordered chains in the cooling process.     

Spectrophotometric titration of phenolic groups of pepsin.

The ionization of tyrosine residues in diazotized pepsin under various solvent conditions was studied. All tyrosyl residues of the protein titrated normally with a pK of 10.02 in 6 M guanidine hydrochloride solution. On the other hand, two stages in the phenolic group titration curve were observed for the inactivated protein in the absence of guanidine hydrochloride; only about 10 tyrosine residues ionized reversibly up to pH 11, above which titration was irreversible. The irreversible titration zone corresponds to the pH range 11--13 in which unfolding, leading to the random coil state, was shown to occur by circular dichroism and viscosity measurements. The number of tyrosine residues exposed in the native and alkali-denatured (pH 7.5) states of diazotized protein were also studied by solvent perturbation techniques; 10 and 12 groups are exposed in the native and denatured states, respectively.     

Deoxydodecanucleotide heteroduplex d(TTTTATAATAAA). d(TTTATTATAAAA) containing the promoter Pribnow sequence TATAAT. I. Double-helix stability by UV spectrophotometry and calorimetry

Thermally induced structural transition in the d(TTTTATAATAAA) d(TTTATTATAAAA) heteroduplex is characterized by UV-spectroscopy and differential scanning calorimetry. At low salt (less than 0.1 M) the occurrence of a cooperative transition in the lower temperature range, followed by a broad transition connected with small increase in absorbance is observed. At high salt (greater than or equal to 0.2 M) a single, monophasic transition appears. Linear dependence of the latter on log of salt concentration (dTm:dlogM = 14.2 degrees C) and of 1/Tm on log of oligomer concentration [derived therefrom delta H (v.H.) = 77.1 kcal/mole (duplex)] allows relating it to the melting of the heteroduplex helix. The non-cooperative transition, independent of oligomer concentration and similar to that of the single chain, was attributed to melting of short hairpin helices upon heteroduplex dissociation. Calorimetric enthalpy: 75.6 kcal/mole (duplex) proved significantly lower than predicted from known calorimetric data for poly[d(AT)] and poly d(A) X poly d(T).     

Synergistic effect of salt mixture on the gelation temperature and morphology of methylcellulose hydrogel

Gelation temperature of methylcellulose (MC) can be altered by adding different additives. Pure MC showed sol-gel transition at 60°C. Sodium citrate and sodium tartrate were used alone and in combination to see the effect of individual salt and combination of salts on the gelation temperature of MC. The gelation temperature of all the binary and ternary combinations of MC and salts were measured with different methods such as test tube tilting method (TTM), UV-vis spectroscopy, viscometry, and by rheometer and also the morphology of gels were characterized with the help of environmental scanning electron microscopy (ESEM). It was observed that when 0.1M sodium citrate (NaC) and 0.1M sodium tartrate (NaT) were used separately, the gelation temperature of MC was reduced up to 44°C and 47°C respectively but when mixture of NaC and NaT (0.1(M) NaC and 0.1(M) (NaT)) were used the gelation temperature was further reduced to 36°C. It was clear from ESEM images that when NaC and NaT were used separately the formation of network was not distinguishable. But, well-connected network structure was observed when a mixture 0.1M NaC and 0.1M NaT was used.     

The pH dependence of the reversible unfolding of ovalbumin A1 by guanidine hydrochloride.

The pH dependence of the reversible guanidine hydrochloride denaturation of the major fraction of ovalbumin (ovalbumin A1) was studied by a viscometric method in the pH range 1-7, at 25 degrees C and at six different denaturant concentrations (1.5-2.6 M). At any denaturant concentrationa reduction in pH favoured the transition from the native to the denatured state. The latter was essentially 'structureless', as revealed by the fact that the reduced viscosity of the acid and guanidine hydrochloride denatured state of ovalbumin A1 (obtained at different denaturant concentrations in acidic solutions) was measured (at a protein concentration of 3.8 mg/ml) to be 29.2 ml/g which is identical to that found in 6 M guanidine hydrochloride wherein the protein behaves as a cross-linked random coil. A quantitative analysis of the results on the pH dependence of the equilibrium constant for the denaturation process showed that on denaturation the intrinsic pK of two carboxyl groups in ovalbumin A1 went up from 3.1 in the native state to 4.4 in the denatured state of the protein.     

NMR evidence for the existence of two native conformations of 5S RNA

NMR spectra of the non-exchangeable protons in 5S RNA from E. coli show the existence of two distinct conformers of the molecule which meet the operational definition of "A form" or native 5S RNA. Both are easily distinguished spectroscopically from denatured, "B form" 5S RNA. The conditions which interconvert the two A form conformers strongly suggest that the transition between them gives rise to the low temperature optical melting transition first reported in 5S RNA by Kao and Crothers (1).     

Differential mobility of skeletal and cardiac tropomyosin on the surface of F-actin.

Polarized phosphorescence from the triplet probe erythrosin-5-iodoacetamide attached to sulfhydryls in rabbit skeletal and cardiac muscle tropomyosin (Tm) was used to measure the microsecond rotational dynamics of these tropomyosins in a complex with F-actin. The steady-state phosphorescence anisotropy of skeletal tropomyosin on F-actin was 0.025 +/- 0.005 at 20 degrees C; the comparable anisotropy for cardiac tropomyosin was 0.010 +/- 0. 003. Measurements of the anisotropy as a function of temperature and solution viscosity (modulated by addition of glycerol) indicated that both skeletal and cardiac tropomyosin undergo complex rotational motions on the surface of F-actin. Models assuming either long axis rotation of a rigid rod or torsional twisting of a flexible rod adequately fit these data; both analyses indicated that cardiac Tm is more mobile than skeletal Tm and that the increased mobility on the surface of F-actin reflected either the rotational motion of a smaller physical unit or the torsional twisting of a less rigid molecule. The binding of myosin heads (S1) to the Tm-F-actin complexes increased the anisotropy to 0.049 +/- 0.004 for skeletal and 0.054 +/- 0.007 for cardiac tropomyosin. The titration of the skeletal tropomyosin-F-actin complex by S1 showed a break at an S1/actin ratio of 0.14; this complex had an anisotropy of 0.040 +/- 0.007, suggesting that one bound head effectively restricted the motion of each skeletal tropomyosin. A similar titration with cardiac tropomyosin reached a plateau at an S1/actin ratio of 0.4, suggesting that 2-3 myosin heads are required to immobilize cardiac Tm. Surface mobility is predicted by structural models of the interaction of tropomyosin with the actin filament while the decrease in tropomyosin mobility upon S1 binding is consistent with current theories for the proposed role of myosin binding in the mechanism of tropomyosin-based regulation of muscle contraction.     

Acid- and base-induced conformational transitions of equinatoxin II

We have investigated the acid- and base-induced conformational transitions of equinatoxin II (EqTxII), a pore-forming protein, by a combination of CD-spectroscopy, ultrasonic velocimetry, high precision densimetry, viscometry, gel electrophoresis, and hemolytic activity assays. Between pH 7 and 2, EqTxII does not exhibit any significant structural changes. Below pH 2, EqTxII undergoes a native-to-partially unfolded transition with a concomitant loss of its rigid tertiary structure and the formation of a non-native secondary structure containing additional alpha-helix. The acid-induced denatured state of EqTxII exhibits a higher intrinsic viscosity and a lower adiabatic compressibility than the native state. Above 50 degrees C, the acid-induced denatured state of EqTxII reversibly denatures to a more unfolded state as judged by the far UV CD spectrum of the protein. At alkaline pH, EqTxII undergoes two base-induced conformational transitions. The first transition occurs between pH 7 and 10 and results in a partial disruption of tertiary structure, while the secondary structure remains largely preserved. The second transition occurs between pH II and 13 and results in the complete loss of tertiary structure and the formation of a non-native, more alpha-helical secondary structure. The acid- and base-induced partially unfolded states of EqTxII form water-soluble oligomers at low salt, while at high salt (> 350 mM NaCl), the acid-induced denatured state precipitates. The hemolytic activity assay shows that the acid- and base-induced denatured states of EqTxII exhibit significantly reduced activity compared to the native state.     

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.