Thermodynamics of conformational transition and chain association of ι-carrageenan in aqueous solution: Calorimetric and chirooptical data

Isothermal microcalorimetry, differential scanning calorimetry (DSC), and chirooptical data obtained for ι-carrageenan in NaCl, LiCl, and NaI aqueous solutions are presented. The experiments have been performed as a function of concentration both for the polymer and for the simple salt as a cosolute. The experimental findings consistently show the occurrence of a salt-induced disorder-to-order transition. From microcalorimetric experiments the exothermic enthalpy of transition ΔH_tr is obtained as the difference between the theoretical, purely electrostatic ΔH_el enthalpy change and the actual mixing enthalpy ΔH_mix, measured when a ι-carrageenan salt-free solution at constant polymer concentration is mixed with a 1:1 electrolyte solution of variable concentration. In the case of added NaCl, the absolute values of enthalpy changes |ΔH_tr| are in good agreement with those obtained for the opposite process, at comparable polymer and salt concentrations, from DSC melting curves. The microcalorimetric results show that the negative maximum value of ΔH_tr corresponding to the interaction of Li⁺ counterion with ι-carrageenan polyion results to be significantly lower than the corresponding values obtained for Na⁺ counterion. At variance with the microcalorimetric data, chirooptical results show that the salt-induced disorder-to-order transition, occurring in the 0.02–0.2M salt concentration range, appears to be complete at a concentration of about 0.08–0.1M of the simple ion, irrespective of the polymer concentration and of the nature of added electrolyte. © 1998 John Wiley & Sons, Inc. Biopoly 45: 105–117, 1998     

A study of the growth of normal and iodinated collagen fibrils in vitro using electron microscope autoradiography

Electron microscopic autoradiographic observations on collagen fibrils grown in vitro allow growth rates in the N- and C-terminal directions to be measured on individual fibrils. Such observations, made on normal and iodinated collagen, show that normal fibrils grow at both ends (although rather more rapidly at the N-terminal end), whereas fully-iodinated collagen fibrils grow only at the N-terminal end. Measurements of growth rates at different temperatures provide estimates of the activation enthalpy (ΔH^≠) and entropy (ΔS^≠) of precipitation for the two types of collagen. Solubility measurements have also yielded values for the thermodynamic enthalpy (ΔH) and entropy (ΔS) of precipitation. Results show that the activated (rate-limiting) state is characterized by a large positive ΔH^≠ and ΔS^≠ similar in magnitude to the ΔH and ΔS of transition from solution to fibril. It is also concluded that the different rates of precipitation of normal and iodinated collagen cannot be explained in terms of fibril formation requiring ionization of the tyrosine residues.     

Temperature-Induced conformational transition in xanthans with partially hydrolyzed side chains

The conformational properties of xanthans with partially hydrolyzed side chains were in vestigated by optical rotation, CD, and differential scanning calorimetry (DSC). All variants displayed the well-known temperature-driven, cooperative order–disorder transition, and both optical rotation and DSC showed that the transition temperature was essentially independent of the content of terminal β-mannose. It was found that up to 80% of the changes in the specific optical rotation accompanying the transition reflects conformational changes linked to the terminal β-mannose in the side chains. Modification of the sidechains also affected the CD when xanthan was in the ordered state, but in this case the data suggest that the glucuronic acid is the major component determining the magnitude of the CD signal. DSC measurements showed that the transition enthalpy (ΔH_cal) increased linearly with the fraction of β-mannose, again indicating that a significant part (up to 80%) of ΔH_cal reflects conformational changes in the side chains. The conformational transition of the xanthan variants generally showed a higher degree of cooperativity (sharper transition) than unmodified, pyruvated xanthan. Calculation of the cooperativity parameter σ by means of the Zimm–Bragg theory (OR data) or from the ratio between ΔH_cal and the van't Hoff enthalpy (ΔH_vH) using DSC data showed a correlation between σ and the content of β-mannose, but the two methods gave different results when the content of β-mannose approached 100%. The ionic strength dependence of the transition temperature, expressed as d (log I)/d(T^?1_m), was nearly identical for intact xanthan and a sample containing only 6% of the terminal β-mannose. Application of the Manning polyelectrolyte theory does not readily account for the observed ΔH_cal values, neither does it provide new information on the nature of the ordered and disordered conformations in xanthan. © 1993 John Wiley & Sons, Inc.     

Thermodynamic analysis of sol–gel transition of gelatin in terms of water activity in various solutions

Sol–gel transition of gelatin was analyzed as a multisite stoichiometric reaction of a gelatin molecule with water and solute molecules. The equilibrium sol–gel transition temperature, T_t, was estimated from the average of gelation and melting temperature measured by differential scanning calorimetry. From T_t and the melting enthalpy, ΔH_sol, the equilibrium sol‐to‐gel ratio was estimated by the van't Hoff equation. The reciprocal form of the Wyman–Tanford equation, which describes the sol‐to‐gel ratio as a function of water activity, was successfully applied to obtain a good linear relationship. From this analysis, the role of water activity on the sol–gel transition of gelatin was clearly explained and the contributions of hydration and solute binding to gelatin molecules were separately discussed in sol–gel transition. The general solution for the free energy for gel‐stabilization in various solutions was obtained as a simple function of solute concentration. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 685–691, 2015.     

Experimental thermodynamics of the helix–random coil transition. II. Calorimetric measurement of transition enthalpies of poly A in acid aqueous solution

The enthalpy change accompanying the double helix–coil transition of polyriboadenylic acid (poly A) in aqueous solution has been measured optically and calorimetrically in the pH range 5.7–4.5. The course of this cooperative transition was followed optically by measuring changes in ultraviolet absorption as a function of temperature at different pH values, and calorimetrically by determining the heat capacity of the solution through the transition region. From the latter measurements, the enthalpy of transition was calculated. It is shown, that ΔH is dependent on pH as it is expected from the influence of protonation of the double helix of poly A.     

Solution properties of synthetic polypeptides. XII. Enthalpy changes accompanying helix-coil transition of polypeptide

For helix-coil transitions of polypeptide in binary mixtures consisting of helix-forming solvent and coil solvent, the transition enthalpy ΔH(T,x) has been found to depend significantly on temperature (T) and solvent composition (x). For such systems, calorimetric measurements may yield some averages of ΔH(T,x) which are no longer amenable to direct comparison with ΔH itself. Theoretical equations relating calorimetric data to ΔH(T,x) are derived and tested favorably with experimental data. It is demonstrated that the transition enthaply from heat capacity measurements is approximately equal to ΔH_cfm, while those from heat of dilution and heat of solution measurements are equal to ΔH_c. Here ΔH_c denotes the value of ΔH at the transition point and f_m represents the maximum helical content attained in a thermally induced transition. The discrepancies among calorimetric data are also discussed.     

Enthalpy of Mixing Glycosaminoglycans with Aqueous NaCl

Calorimetric measurements of ΔH , the enthalpy of mixing with NaCl solutions per mole of polymer charge equivalent, have been made for the sodium salts of hyaluronate, chondroitin 4-sulfate, and chondroitin over the range m_s^f = 0–0.5 (where m_s^f is final NaCl molality); corrections were made for the enthalpy of dilution of NaCl. Values of ΔH for the range m_s^f = 0–0.1 agree reasonably well with a theoretical estimate from the infinite line charge theory of polyelectrolytes. In order to apply the latter, calculations of several average short-range atomic spacings in hyaluronate were made from a statistical model with use of energy parameters which provide a good fit to experimental values of unperturbed dimensions.     

Effect of hydration upon the thermal stability of tropocollagen and its dependence on the presence of neutral salts

The endotherm enthalpy changes ΔH_D and temperatures T_D of thermal denaturation of tropocollagen fibers were measured by DSC calorimetry as functions of water content. The denaturation temperatures decrease with increasing water content. The enthalpy change values increase sharply in the range 0–28% of water content, where a maximum of 14.3 cal g^?1 is reached. The effect of water uptake on the enthalpy term is explained by water bridge formation within the collagen triple helix. Evidence is given for the existence of approximately three intercatenary water bridges per triplet at the enthalpy maximum, their H-bond energy amounting to approximately 4000 kcal/mol of protein. In the 30–60% range of water content, ΔH_D decreases by 2 cal^?1 probably due to interactions between secondary water structures and the stabilizing intrahelical water bonds. The influence of two neutral potassium salts, with a structure-stabilizing and a structure-breaking anion (F^? and I^?), on the hydration dependence of ΔH_D and T_D was also studied. It was shown that the primary hydration is not influenced by these ions, but that T_D and ΔH_D are altered in an ion specific way in the presence of interface and bulk water. Hydrophobic interactions do not explain the experimental results. A reaction mechanism of the effects of ions upon the structural stability of collagen is proposed and discussed in terms of interactions of the medium water molecules with the intrahelical water bonds, and in terms of proton-donor/proton-acceptor equilibria between peptide groups, hydrated ions, and intrahelical water molecules.     

Volume and sound velocity changes associated with coil- transition of poly(S-carboxymethyl L-cysteine) in aqueous solution

The measurements were made for the volume and the sound velocity changes (ΔV and ΔU) on titrating the sodium salt of poly (S-carboxymethyl L -cysteine) with dilute HCl. For the reaction, ? COO^? + H⁺ → ? COOH, ΔV per mole of H⁺ bound was + 12. 7 ml and +11. 4 ml in salt-free and 0. 2 M NaCl solutions, respectively. Corresponding ΔU was about ?13 cm/sec in salt-free polymer solution where 11.5 mM carboxylate ion reacts with equimolar hydrogen ion. ΔV associated with the coil-to-β transition was found to be +2. 35 ml in H₂O and +1. 90 ml in 0. 2 M NaCl per mole of amino acid residue, respectively. These values are larger than those obtained for the coil-to-helix transition of poly (L -glutamic acid). ΔU for the transition was about ?30 cm/sec in salt-free solution of polymer concentration 0.0115 mole/liter. Possible sources of ΔV and ΔU for reaction; coil → β, are (1) the formation of void volume and (2) the changes in the extent of solvation in amide linkage and in side chain.     

Experimental theromodynamics of the helix-random coil transition. I. Influence of polymer concentration and solvent composition in the PBG-DCA-EDC system

The course of the reversible helix formation of poly(γ-benzyl L -glutamate) (PBG) dissolved in a mixture of dichloroacetic acid (DCA) and 1,2-dichloroethane (EDC) was followed by measuring the heat capacity and the optical rotation of the system through the transition region. The results of these measurements indicate that the transition enthalpy ΔH the transition temperature T_c, and the Zimm-Bragg parameter σ depend considerably on the PBG concentration as well as on the composition of the solvent. For the standard state of infinite dilution, however, a linear extrapolation of the measured ΔH if values results in a standard value ΔH° = 950 cal./mole, independent of the solvent composition. The results of the calorimetric measurements are discussed in relationship to changes in optical rotation. Some peculiarities in the measured thermodynamic and optical properties in solutions with relatively high content of dichloroacetic acid are reported.     

Thermodynamics of the B to Z transition in poly(dGdC)

The thermodynamics of the B to Z transition in poly(dGdC) was examined by differential scanning calorimetry, temperature-dependent absorbance spectroscopy, and CD spectroscopy. In a buffer containing 1 mM Na cacodylate, 1 mM MgCl₂, pH 6.3, the B to Z transition is centered at 76.4°C, and is characterized by ΔH_cal = 2.02 kcal (mol base pair)^?1 and a cooperative unit of 150 base pairs (bp). The t_m of this transition is independent of both polynucleotide and Mg²⁺ concentrations. A second transition, with ΔH_cal = 2.90 cal (mol bp)^?1, follows the B to Z conversion, the t_m of which is dependent upon both the polynucleotide and the Mg²⁺ concentrations. Turbidity changes are concomitant with the second transition, indicative of DNA aggregation. CD spectra recorded at a temperature above the second transition are similar to those reported for ψ(–)-DNA. Both the B to Z transition and the aggregation reaction are fully and rapidly reversible in calorimetric experiments. The helix to coil transition under these solution conditions is centered at 126°C, and is characterized by ΔH_cal = 12.4 kcal (mol bp)^?1 and a cooperative unit of 290 bp. In 5 mM MgCl₂, a single transition is seen centered at 75.5°C, characterized by ΔH_cal = 2.82 kcal (mol bp)^?1 and a cooperative unit of 430 bp. This transition is not readily reversible in calorimetric experiments. Changes in turbidity are coincident with the transition, and CD spectra at a temperature just above the transition are characteristic of ψ(–)-DNA. A transition at 124.9°C is seen under these solution conditions, with ΔH_cal = 10.0 kcal (mol bp)^?1 and which requires a complex three-step reaction mechanism to approximate the experimental excess heat capacity curve. Our results provide a direct measure of the thermodynamics of the B to Z transition, and indicate that Z-DNA is an intermediate in the formation of the ψ-(–) aggregate under these solution conditions.     

Effects of sugars and polyols on the sol-gel transition of k-carrageenan: calorimetric study

The effects of sugars and polyols on the sol-gel transition of k-carrageenan were studied in 0.025 m KCl by means of differential scanning calorimetry. Addition of these compounds invariably raised the gelling temperature T_g, with an increase in their concentration, accompanying a decreased (less negative) enthalpy of gelation, ΔH_g. This indicates that it is not ΔH_g but the entropy of gelatin, ΔS_g, which plays an essential role in the gel stabilizatiion by them, thus differing from the enthalpy-driven stabilization by addition by KCl, ethanol or carrageenan. It seemed that such a large change in ΔS_g relative to ΔH_g predominantly occurs in the process of replacing polymer solvent hydrogen bonds by polymer—polymer hydrogen bonds with minor contribution of the conformational entropy of polymer chains. The gel-stabilizing ability of different sugars and polyols is discussed in terms of their different influences on the structure of water.     

Stabilization of collagen structure: Dependence of collagen denaturation enthalpy on the imino acid content

An analysis of the available data on the enthalpy (ΔH_r) of denaturation (melting) of collagens with different imino acid content in solution and in the aggregated state has shown that ΔH_r in solution increases with increasing denaturation temperature, whereas in the aggregated state there is an inverse dependence. ΔH_r in solution correlates with the hydroxyproline content but not with that of proline. No correlation between the change of ΔH_r and the imino acid content is observed for the aggregated state.     

Thermodynamic parameters of helix-coil transition in polypeptide chains. II. Poly-L-lysine

The helix–coil transitions for poly-L -lysine (PL) were investigated by the methods of spectropolarimetry, viscometry and potentiometric titration in 0.2M NaCl at different temperatures as well as in 0.2MNaBr, 1MKCl, and in mixtures of 0.2MNaCl or NaBr with methanol at room temperature. The enthalpy and entropy differences between the helical and coillike states of uncharged PL molecules in 0.2.M NaCl were determined from the potentiometric titration curves. The cooperativity parameters σ for PL in different solvents were determined by two methods (from the sharpness of the transition and from the dependence of the intrinsic viscosity on the helical content in the transition region). In 0.2MNaCl σ has a value of (2.3 ± 0.5) × 10^?4 and does not depend on temperature, i.e., the cooperativity of the helix-coil transition, as for PGA, is mainly of an entropy origin (the initiating of the helical region is accompanied by the entropy decrease ΔS_i = ?12 eu/mole of helical regions). A comparison of the obtained results for PGA and PL with the molecular theories of the helix-coil transitions shows that the role of dipole-dipole interactions of nonneighboring peptide groups is greatly overestimated in these theories, leading to a considerable enthalpy contribution to the free energy of initiating helical regions which is not observed in the experiment.     

Solvent isotope effect on the differences in structure and stability between normal and deuterated proteins

Differential scanning microcalorimetry was used to investigate the enthalpy (ΔH_d) and the temperature (t_d) of thermal denaturation of normal (nondeuterated) (H-PC) and deuterated (D-PC) phycocyanins in D₂O solvent. Values of t_d in D-PC are about 5–7°C lower than those in H-PC. The magnitudes of ΔH_d in D-PC are only 21–32% of those in H-PC. During the protein unfolding, the heat-capacity changes (ΔC_p) in D-PC are also lower than those in H-PC. CD was employed to evaluate the secondary structure and the urea denaturation of these proteins in D₂O solvent. These proteins have about the same α-helix content. D-PC is less resistant to the denaturant urea than is H-PC. In general, the apparent free-energy change in the process of protein unfolding at zero denaturant concentration is higher in H-PC than in D-PC. Comparisons of the present results for D₂O solvent with those previously reported for H₂O reveal that solvent isotope effect essentially does not change the α-helix content in H-PC and D-PC. However, D-PC or H-PC has a higher random-coil content in its secondary structure in D₂O than in H₂O. Substitution of H₂O with D₂O as the solvent increases t_d in both D-PC and H-PC, lowers ΔH_d in H-PC, and greatly lowers ΔH_d in D-PC. The deuterium solvent isotope effect does not change ΔC_p in H-PC but lowers ΔC_p in D-PC. In the urea denaturation, the magnitudes of (C_u)_1/2 in H-PC and D-PC are not affected by such a solvent effect, whereas those of ΔG are greatly increased. These results are correlated with the structure and stability of the proteins.     

Nucleic acid-solvent interactions: temperature dependence of the heat of solution of thymine in water and ethanol

The heats of solution of thymine in water and ethanol have been determined calorimetrically as a function of temperature. These data, along with solubility data, have been used to calculate the thermodynamic quantities (ΔG_t, ΔH_t, ΔS_t and ΔC_p,t) associated with the transfer of thymine from ethanol to water. Since ΔS_t = ?2 cal/mole deg and ΔC_p,t = 0, it has been concluded that hydrophobic bonding does not play an important role in the thermocynamic stability of nucleic acids. However, large heat capacities of solution of thymine are observed in both solvents (ΔC°_p2 = 45 ± 4 cal/mole deg). This is explained in terms of temperature variation in the degree of solvent–solute hydrogen bonding. It is our proposal that the components of macromolecules (i.e., nucleic acid bases and amino acids) do not make all possible hydrogen bonds with the solvent in the vicinity of room temperature. Thus the thermodynamic contribution of hydrogen bonding to the stability of macromolecules in aqueous solution must be reassessed.     

Chain length and oligonucleotide stability at high pressure

The effect of hydrostatic pressure on the helix-coil transition temperature (T_m) was measured for the DNA oligomers (dA)_n(dT)_n, where n = 11, 15, and 19, in 50 mM NaCl. The data were analyzed in light of previously published data for the polymer, poly(dA)·poly(dT) under the same conditions. As has been observed for DNA polymers, increasing the hydrostatic pressure led to an increase in the T_m of the oligomers; however, the effect of pressure diminished with decreasing chain length. The value of dT_m/dP decreased linearly with the inverse of the chain length varying from 3.15 × 10⁻²°C MPa⁻¹ for the polymer to 0.7 × 10⁻²°C MPa⁻¹ for the 11-mer. The two-state or van't Hoff enthalpy (ΔH_vH) of the helix-coil transition was obtained by analysis of the half-width of the thermal transition. As expected, ΔH_vH decreases with decreasing chain length. In contrast to the behavior of the polymer, poly(dA)·poly(dT), and (dA)₁₉(dT)₁₉, the ΔH_vH of the two shorter duplex oligonucleotides displayed a small pressure dependence dΔH_vH/dP≃−0.4 kJ MPa⁻¹ in both cases. The changes observed in the T_m and ΔH_vH were not sufficient to explain the magnitude of the chain-length dependence of the pressure effect. To interpret the large chain-length dependence of dT_m/dP, we propose that the terminal base pairs contribute a negative volume change to the helix-coil transition. Base pairs distant from the ends exhibit behavior characterized by the polymer where end effects are assumed to be negligible, i.e., a positive volume change for the helix-coil transition. The negative volume change of separating terminal bases may originate from the imperfect interactions these base pairs form with water due to the existence of several energetically equivalent conformations. This is reminiscent of one of the mechanisms proposed to be important in the pressure-induced dissociation of multimeric proteins into their constituent subunits. © 1996 John Wiley & Sons, Inc.     

Calorimetric and spectroscopic investigation of the helix-to-coil transition of the self-complementary deoxyribonucleotide ATGCAT

Differential scanning calorimetry and temperature-dependent uv spectroscopy are used to thermodynamically characterize the double-strand to single-strand transition of the self-complementary deoxyribo-oligonucleotide ATGCAT. The calorimetric experiments provide a value of 33.6 kcal (mol of double strand)^?1 for the transition between 10 and 90° C. In conjunction with available temperature-dependent nmr data (which reveals terminal base pair fraying), we attempt to define specifically those interactions to which the calorimetrically measured enthalpy change refers.Values of ΔH_V.H. (van 't Hoff enthalpy change) are derived from the spectroscopic and calorimetric data and compared with the ΔH obtained directly from the calorimetric experiment. This comparison reveals that the part of the thermally-induced transition that occurs between 10 and 90°C is well represented by a two-state process. It is noted that in assessing the applicability of the two-state model it is best to compare the ΔH_cal. with ΔH_V.H. obtained from the calorimetric rather than the spectroscopic data.     

Large contributions of coupled protonation equilibria to the observed enthalpy and heat capacity changes for ssDNA binding to Escherichia coli SSB protein

Many macromolecular interactions, including protein‐nucleic acid interactions, are accompanied by a substantial negative heat capacity change, the molecular origins of which have generated substantial interest. We have shown previously that temperature‐dependent unstacking of the bases within oligo(dA) upon binding to the Escherichia coli SSB tetramer dominates the binding enthalpy, ΔH_obs, and accounts for as much as a half of the observed heat capacity change, ΔC_p. However, there is still a substantial ΔC_p associated with SSB binding to ssDNA, such as oligo(dT), that does not undergo substantial base stacking. In an attempt to determine the origins of this heat capacity change, we have examined by isothermal titration calorimetry (ITC) the equilibrium binding of dT(pT)₃₄ to SSB over a broad pH range (pH 5.0–10.0) at 0.02 M, 0.2 M NaCl and 1 M NaCl (25°C), and as a function of temperature at pH 8.1. A net protonation of the SSB protein occurs upon dT(pT)₃₄ binding over this entire pH range, with contributions from at least three sets of protonation sites (pK_a1 = 5.9–6.6, pK_a2 = 8.2–8.4, and pK_a3 = 10.2–10.3) and these protonation equilibria contribute substantially to the observed ΔH and ΔC_p for the SSB‐dT(pT)₃₄ interaction. The contribution of this coupled protonation (∼ −260 to −320 cal mol⁻¹ K⁻¹) accounts for as much as half of the total ΔC_p. The values of the “intrinsic” ΔC_p,0 range from −210 ± 33 cal mol⁻¹ °K⁻¹ to −237 ± 36 cal mol⁻¹K⁻¹, independent of [NaCl]. These results indicate that the coupling of a temperature‐dependent protonation equilibria to a macromolecular interaction can result in a large negative ΔC_p, and this finding needs to be considered in interpretations of the molecular origins of heat capacity changes associated with ligand‐macromolecular interactions, as well as protein folding. Proteins 2000;Suppl 4:8–22. © 2000 Wiley‐Liss, Inc.