Polypeptides. 53. Water-soluble copolypeptides of L-glutamic acid, L-lysine, and L-alanine

The synthesis and some of the physical-chemical properties of tricopolymers of L -glutamic acid, L -lysine, and L -alanine are reported here. The molar ratios of the glutamyl: lysyl: alanyl residues were 1:1:X or 3:2:X, where the alanyl content X was increased in regular steps. The α-helix content calculated from the optical rotatory dispersion of the polypeptides is compared with a predicted helix content estimated from the composition of the polymers and the known behavior of the homopolypeptides at pH 3, 8, and 12. At pH 3 copolypeptides containing 20 mole-% or more alanine exhibit a helix content equal to the sum of their alanyl and glutamyl residue contents. At pH 8 the helix content equals the alanyl content when the latter was 40 mole-% or higher; at lower alanyl contents the electrostatic interaction between charged glutamyl and lysyl residues makes some contribution. At pH 12 the amount of helix observed is proportional to the mole ratio of alanine residues present in the polymer. The helix content of a tricopolymer containing 1:1:3 mole ratios of glutamyl: lysyl: alanyl residues was determined in solutions of lithium bromide and in urea solutions. Both reagents led to a decrease in helix content at pH 3 and 8 to a minimum of approximately 20% helix in 8M urea or 5.5M LiBr. The helix–random chain transition curves at pH 3 and 8 are parallel when the urea concentration is varied, but differ in shape when the lithium bromide concentration is varied at pH 3 and 8. The mode of action of these two “denaturing” reagents may thus be different. Heating the same tricopolypeptide at pH 3 or 8 from 5 to 80°C. also led to a helix–random chain transition centered at approximately 45°C.     

Calorimetric measurement of enthalpy change in the isothermal helix--coil transition of poly-L-lysine in aqueous solution

The heat ΔH° for converting an uncharged lysine residue from a coil to an α-helical state in poly-L -lysine in 0.1N KCl has been determined calorimetrically to be ?1200 cal/mole at both 15°C and 25°C. Essentially the same value has been obtained for the conversion of an uncharged residue from a coil to a β-pleated sheet state. Titration data provided information about the state of charge of the polymer in the calorimetric experiments, and optical rotatory dispersion data about its conformation. In order to compute ΔH°, the observed Calorimetric heat was corrected for the heat of breaking the sample cell, the heal of dilution of HCl, the heat of neutralization of OH^? ion, and the heat of ionization of the ε-amino group in the random coil. The latter was obtained from similar Calorimetric measurements on poly-D ,L -lysine, which was shown to be a good model for the random coil form of poly-L -lysine. The measured transition heat was ～0.7 cal., which is only 7% of the total heat liberated when a 40 ml solution of 0.25% w/v poly-L -lysine is brought, from pH 11 to pH 7; nevertheless it could be determined with a precision of ±8%. The conformation of poly-L -lysine at pH 11 appears to be completely helical at 15°C, but a mixture of 90% α-helix, 5% β form, and 5% coil at 25°C. Since ΔH° ～ 0 for the α ? β conversion, the polymer behaves like one of 95% α-helix and 5% coil in the calorimeter at 25°C. At neutral pH, poly-L -lysine is an extended coil, like poly-D ,L -lysine.     

Conformational studies of poly-L-alanine in water

The conformational properties of poly-L -alanine have been examined in aqueous solutions in order to investigate the influence of hydrophobic interactions on the helix–random coil transition. Since water is a poor solvent for poly-L -alanine, water-soluble copolymers of the type (D , L -lysine)_m–(L alanine)_n-(D , L -lysine)_m, having 10, 160, 450, and 1000 alanyl residues, respectively, in the central block, were synthezised. The optical rotatory dispersion of the samples was investigated in the range 190–500 mμ, and the rotation at 231 mμ was related to the α-helix content, θ_H, of the alanine section. In salt-free solutions, at neutral pH, the three large polymers show high θ_H values, which are greatly reduced when the temperature is increased from 5 to 80°C. No helicity was observed for the small (n = 10) polymer. By applying the Lifson-Roig theory, the following parameters were obtained for the transition of a residue from a coil to a helical state: ν = 0.012; ΔH = ?190 ± 40 cal./mole; ΔS = ?0.55 ± 0.12 e.u. Since ΔH and ΔS differ from the values expected for a process involving only the formation of a hydrogen bond, and in a manner predicted by theories for the influence of hydrophobic bonding on helix stability, it is concluded that a hydrophobic interaction is also involved. In the presence of salt (0.2M NaCl), or when the ε-amino groups of the lysyl residues are not protonated (pH = 12), the helical form of the two large polymers (n = 450 and n = 1000) is more stable than in water. Since the electrostatic repulsion between the lysine end blocks is greatly reduced under these conditions, the alanine helical sections fold back on themselves, and this conformation is stabilized by interchain hydrophobia bonds. This structure was predicted by the theory for the equilibrium between such interacting helices, non-interacting helices, and the random coil.     

Conformational studies on copolymers of L-lysine and L-leucine: circular dichroism and potentiometric titration studies

Conformational aspects of a series of copolymers of L -Leucine and L -leucine [poly-(Lys^xLeu^y)] containing 0 to 0.41 mole fraction L -leucine have been studied by circular dichroism (CD) and potentiometric titration in 0.05M KF solution. CD studies on the α-helical conformation showed a dependence of the magnitude of the CD ellipticity band at 222 nm on copolymer composition; the [θ]₂₂₂ decreasing with higher leucine contents. This was interpreted as the result of an increase of the hydrophobicity of the environment of the amide group due to the presence of the leucyl residues. Values of the Zimm-Rice parameter, σ, for the copolymers were obtained from the potentiometric titrations and used to fit theoretical curves to the experimental data. Using the variation of σ with polymer composition, a value of σ for the leucyl residue was estimated to be 6.3 × 10^?2, assuming independence of σ on the amino acid sequence in the copolymer. The free energy change for the conversion of one mole residue from uncharged helix to uncharged coil, ΔG_hc°, was also obtained from the titration data for each copolymer up to a leucine mole fraction of 0.16; a value of 385 cal mole^?1 was estimated for ΔG_hc° for a leucyl residue. These values for σ and ΔG_hc° are compared with other values in the literature for various amino acid residues obtained from titration and melting curve data.     

Thermodynamic parameters for the helix-coil thermal transition of ribonuclease-S-peptide and derivatives from 1H-NMR data

Values for the thermodynamic quantities, ΔH° = 11.8 ± 2.0 Kcal/mole and ΔS° = 43.6 ± 6.0 e.u., of the 3-13 helix–coil equilibrium of isolated S-peptide (19 residue N-terminal fragment of ribonuclease A) in aqueous solution (3 m M, 1M NaCl, pD 5.4) have been determined from a joint analysis of the Thr 3γ, Ala 6β, Phe 8meta, and Phe 8para ¹H chemical shift vs temperature curves (?7 to 80°C) in several aqueous–trifluorethanol mixtures. Chemical shifts in the coil and in the helix have been determined for up to 16 protons belonging to the 3-13 fragment. Thermodynamic parameters have also been determined for C-peptide (13 residue fragment) and a number of S-peptide derivatives. From the variation of the values of the thermodynamic parameters at pD 2.5, 5.4, and 8.0, a quantitation of the two helix-stabilizing side-chain interactions can be made: (1) Δ(ΔH°) ? 5 Kcal/mole and Δ(ΔS°) ? 18 e.u. for the salt bridge Glu 2^? … Arg 10⁺ and (2) Δ(ΔH°) ? 3 Kcal/mole and Δ(ΔS°) = 9 e.u. for the one in which the His 12⁺ imidazolium group is involved, presumably a partial stacking with the Phe 8 side chain.     

Helix-coil transition of Poly(L-glutamic acid) in N-methylacetamide

The folding of randomly coiled poly(L -glutamic acid) to the helical state has been studied in N-methylacetamide by titration methods. Since this solvent would be expected to form amide-peptide group hydrogen bonds with the unfolded form of the polymer, to a first approximation no helix stabilization could come from intrapolymer hydrogen bonds. The titration data, collected from 30 to 70°C yield the following values per residue for the thermodynamic parameters governing the coil-helix reaction for the uncharged polymer: ΔG_30°C°, ?1. 9 ± 0.1 kcal; Δ H°, 0 ± 0.1 kcal; ΔS_30°C°, 6.3 ± 0.6 eu. In N-methyl acetamide, the helix is an order of magnitude more stable than in water, and this stabilization appears to be entirely the result of the entropy gained by solvent molecules which are released from the polymer upon folding.     

Cis-Trans equilibrium and kinetic studies of acetyl-L-proline and glycyl-L-proline

By means of carbon-13 nmr (at 25 MHz) the trans/cis conformer ratio in glycyl-L -proline has been measured in aqueous (D₂O) solution over the temperature range 33–96°C. It is found that ΔH⁰ = ?4.2 kJ/mole and ΔS⁰ = ?9.7 J/mole/K. Measurements of the T₁ values for the proline ring carbons yielded values consistent with a fast puckering process involving both the β- and γ-carbons. Measurements of the rate of cis-trans conformational interconversion in glycyl-L -proline, using complete line-shape analysis for the glycyl α-carbon resonance, gave values for the trans → cis isomerization as follows: ΔH^≠ = 83.5 ± 0.2 kJ/mole; ΔS^≠ = 0.0 ± 10 J/mole/K. A more approximate determination from coalescence temperature observations gave a value of ΔG^≠ of 82.0 ± 0.4 kJ/mole for this process in acetyl-L -proline in aqueous solution. The presence of 12M NaSCN lowered this barrier by ca. 2.6 kJ/mole. Such measurements are relevant to present theoretical models of the denaturation-renaturation processes in proteins, in which proline residues may play a key role.     

The effect of neutral salts on the conformational transition of poly-alpha-amino acids. I. Potentiometric titration and optical rotatory dispersion studies

The effect of salts on the coil-to-helix transition of poly-α-amino acids was investigated by optical rotatory dispersion and potentiometric titration techniques. Both charge-dependent and charge-independent contributions to the free energy were considered. The free energy of formation ΔF° of the uncharged α-helix from the uncharged random coil for poly-L -glutamic acid (PGA) decreases very rapidly in the limit of zero added salt concentration. This effect probably depends on the uncertainty affecting the choice of the extrapolation of the apparent pK for the random coil at low ionic strength. Above 0.1 M salt, where the free energy determination becomes meaningful, the anions and cations investigated do not affect the value of ΔF°, with the exception of Li⁺. Our data support the point of view that this cation binds to the peptide group. A class of salts produces an increase of the helical content of poly-L -ornithine (PO) both at low and high degree of ionization. This effect appears to be anion dependent. It is shown that (1) no change of ΔF° is involved; (2) recent theories of polyelectrolyte solutions cannot account for our results. We suggest that a true site binding of the anions to the charged amino groups occurs. The role of electrostatic binding in determining the conformational stability of proteins in the presence of some anions is stressed, and a general treatment for the electrostatic binding equilibria is outlined.     

The random coil leads to beta transition of copolymers of L-lysine and L-valine: potentiometric titration and circular dichroism studies.

A series of copolymers of L -lysine and L -valine [poly(L -lysine^f L -valine^100-f)] containing 0–13% L -valine have been studied, in 0.10M KF solution, using potentiometric titration and circular dichroism spectroscopy. Incorporation of increasing amounts of valine into the copolymers favors β-sheet formation over α-helix formation at high pH and room temperature. The titrations were analyzed using the method of Zimm and Rice and the partial free energy (ΔG⁰_cβ) for the coil-to-β-sheet transition for valine is estimated at 900 cal/mole at 25°C. From the temperature dependence of the free energy, the partial enthalpy, ΔH⁰_cβ, and entropy, ΔS⁰_cβ, of the transition for valine is estimated to be 854 cal/mole and 6.0 e.u., respectively. The corresponding partial thermodynamic parameters for L -lysine are in agreement with published results. The fraction of β-sheet versus pH has been calculated for poly(L -lysine^86.8 L -valine^13.2) at 25.0°C using the titration data; data obtained from circular dichroism spectroscopy for the same copolymer are in good accord. It is concluded from these results that L -valine is a very strong β-sheet forming amino acid. Furthermore, these results indicate that the Zimm–Rice method is applicable to transitions between the coil and β-sheet states for a polypeptide containing two different residues.     

Thermodynamic parameters of helix-coil transition in polypeptide chains. I. Poly-(L-glutamic acid)

The helix-coil transitions for poly(L -glutamic acid) (PGA) in 0.2M NaCl and in its mixture with dioxane were studied by the methods of spectropolarimetry, viscometry, and potentiometric titration at different temperatures from 8 to 50°C. The enthalpy and entropy differences between the helical and coillike states of uncharged PGA molecules were determined from the curves of potentiometric titration. The temperature dependence of the cooperativity parameter σ was determined by two methods: from the sharpness of transition and from the dependence of the intrinsic viscosity on the helical content in the transition region. In 0.2MNaCl, σ= (2.5 ± 0.5) × 10^?3 and practically does not depend on temperature, i.e., the cooperativity of the helix-coil transition is connected mainly with the entropy decrease in initiating helical regions (ΔS_i ≈ ?12 is mole of helical regions). On the contrary, initiation of a helical region in the water-organic solvent mixture is accompanied by a considerable enthalpy increase.     

Volume and sound velocity changes accompanying the -helix to -form and coil to -helix transitions in aqueous solution

The volume increment per amino acid residue for the α-helix to β-form transition of uncharged poly-L -lysine in aqueous solution was 3.8 ml in water and 4.3 ml in 0.2M and 1M NaBr solutions at 26°C, respectively. The sound velocity of the polymer solution was greater with the β-helix than with the β-form, but the difference was less in dilute salt solutions and disappeared in 1 or 2M NaBr solution. Thus, the β poly-L -lysine solution was slightly more compressible than the α-polymer solution, but this difference was diminished with increasing salt concentration. Both the volume change and the change in adiabatic compressibility of the polymer solution suggest that hydrophobic interactions among the lysyl groups in the β-form reduce the amount of “icebergs” surrounding the polymer molecules as compared with the amount originally present with the α-helix. The coil-to-helix transition of poly-L -glutamic acid in aqueous solution was also accompanied by a decrease in sound velocity. This can be attributed to the reduction of the water of hydration which is less compressible than free water.     

Stability of the helical conformation of random L-alanine-L-lysine copolymers in aqueous solution

The potentiometric titration of random copolymers of L -lysine and L -alanine containing 0–35% alanine was carried out. The standard free-energy change for the transition of coil to helix was calculated from the titration curve, and was treated by taking account of first-neighbor interactions. For uncharged lysine ΔG° = ?140 cal/mole, and for alanine ΔG° = ?50 cal/mole in 0.06M NaBr at 25°C, indicating that the alanine helix is thermodynamically less stable than the lysine helix. The randomness in co-polymerization was confirmed by trypsin treatment.     

Stability of polypeptide conformational states as determined by computer simulation of the free energy

A method is developed to extract the entropy of polypeptides and proteins from samples of conformations. It is based on techniques suggested previously by Meirovitch, and has the advantage that it can be applied not only to states in which the molecule undergoes harmonic or quasiharmonic conformational fluctuations, but also to the random coil, as well as to mixtures of these extreme states. In order to confine the search to a region of conformational space corresponding to a stable state, the transition probabilities are determined not by “looking to the future,” as in the previous method [H. Meirovitch and H. A. Scheraga (1986) J. Chem. Phys. 84 , 6369–6375], but by analyzing the previous steps in the generation of the chain. The method is applied to a model of decaglycine with rigid geometry, using the potential energy function ECEPP (Empirical Conformational Energy Program for Peptides). The model is simulated with the Metropolis Monte Carlo method to generate samples of conformations in the α-helical and hairpin regions, respectively, at T = 100 K. For the α-helix, the four dihedral angles of the N- and C-terminal residues are found to undergo full rotational variation. The results show that the α-helix is a more stable structure than the hairpin. Both its Helmholtz free energy F and energy E are lower than those of the hairpin by ΔF ～ 0.4 and ΔE ～ 0.3 kcal/mole/residue, respectively. It should be noted that the contribution of the entropy ΔS to ΔF is significant (TΔS ～ 0.1 kcal/mole/residue). Also, the entropy of the α-helix is found to be larger than that of the hairpin. This is a result of the extra entropy arising from the rotational freedom about the four terminal single bonds of the α-helix.     

Volume changes accompanying the complex formation of polyadenylic and polyuridylic acids

The complex formation of polyadenylic acid (poly A) and polyuridylic acid (poly U) in 0.1M NaCl solution containing 0.01M sodium cacodylate was followed by dilatometric measurements at various mixing ratios of poly A and poly U. The volume changes, ΔV, accompanying the formation of poly A. poly U and poly A.2poly U were + l.5 and + 2.5 ml per mole of the nucleotide residue, respectively. This increase in volume was probably due to the increased counterion binding when the single-stranded polynucleotides were converted into the double- and triple-stranded helices, since depletion of charged species from the solvent proper would lessen the effect of electrostriction, thus resulting in a positive ΔV. The conversion of a single-stranded poly A to a double-stranded helix in acidic solution led to a ΔV of + 3.8 ml per mole of the nucleotide residue. This increase in volume was attributed to the charge neutralization as a result of protonation of the adenine bases.     

Helix-coil transition of poly-gamma-N-carbobenzoxy-L-alpha, gamma-diaminobutyrate and poly-delta-N-carbobenzoxy-L-ornithine

The helix–coil transition of poly-γ-N-carbobenzoxy-L -α,γ-diaminobutyrate (PCLB) and poly-δ-N-carbobenzoxy-L -ornithine (PCLO) in chloroform–dichloroacetic acid mixtures was followed by optical rotatory dispersion. PCLB displays a “normal” temperature-induced transition, but PCLO an “inverse” one. The thermodynamic parameters for helix formation of the two polymers were determined using the Zimm-Bragg theory. The enthalpy for adding an amide residue to a helical region, ΔH, and the initiation factor σ were ΔH = ?180 cal/mole and σ = 9.2 × 10^?5 for PCLB and ΔH = +490 cal/mole and σ = 1.9 × 10^?5 for PCLO.     

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.     

Basic polypeptides as histone models. Synthesis, conformation, and interaction with DNA of statistical copolymers (Lys x,Ala y)n.

Statistical copolymers (Lys^x,Ala^y)_n were synthesized by copolymerization of N-carboxyanhydrides of L -amino acids. The conformation of copolymers in aqueous solutions was investigated using circular dichroism (CD). Calculations based on the CD data showed that polymers (Lys^x,Ala^y)_n can exhibit a random conformation, an α-helix, and a β-structure in various ratios. CD spectra of complexes of copolymers with DNA prepared by gradual dialysis from a high ionic strength to 0.15 M NaCl can be correlated with the copolymer conformation in medium and high ionic strength. For copolymers forming an α-helix and β-structure, these spectra show resemblance with similar spectra of complexes of those histones that are able to exhibit ordered conformations.     

CD studies of synthetic polypeptides as histone models: Poly(L-Arg-L-Ile-Gly), poly(L-Arg-L-Nva-Gly), and poly(L-Arg-L-Nle-Gly)

Sequential polypeptides (L -Arg-X-Gly)_n were prepared as synthetic models of arginine-rich histones to study their structure and their stereospecific interactions with DNA. In our previous work the conformational characteristics of poly(L -Arg-L -Ala-Gly), poly(L -Arg-L -Val-Gly), and poly(L -Arg-L -Leu-Gly) have already been analyzed. To obtain further insight into the influence of the X residue side chain on the conformation of the (L -Arg-X-Gly)_n polytripeptides, we now report their synthesis and cd properties when X represents the amino acid residues Ile, Nva, and Nle. The pentachlorophenyl active esters of the appropriate tripeptides were used to perform the polymerization, and the toluene-4-sulfonyl group was used to protect the arginine guanido group. CD spectroscopy showed that, in 100% trifluoroethanol, the degree of helical conformation increased in the order Ile → Nle → Nva. An equilibrium between β-turn, α-helix, and random-coil conformers occurred in 100% hexafluoroisopropyl alcohol, while a rise in the temperature or the addition of water favored the α-helix, the highest percentage of which was observed in a mixture of hexafluoroisopropyl alcohol: water (20 : 80) and in the order Ile → Nle → Nva. In aqueous solutions (at pH 7 and 12) the polymers behaved as a random coil, but they were forced to a less aperiodic structure, over a range of ionic strengths (0–0.5M NaF). A rise in temperature of up to 70°C in 100% trifluoroethanol resulted in a decrease of the α-helix percentage of the polymers, while in aqueous solutions the aperiodic structure decreased with increasing temperature. This study proved the importance of the nature of the X residue (length, C_β branching) in relation to the structural order of the sequential polypeptides. We concluded that the polymers prepared are suitable models for arginine-rich histones.     

Conformation of poly(L-arginine). II. Complexes with polyanions

Conformaitons of poly(L -arginine)/polyanion complexes were studies by CD measurements. The polyanions were the homoplolypeptides poly(L -glutamic acid) and poly(L -aspartic acid); the synthetic polyelectrolytes and polyethylenesulfonate; and the polynucleotides were native DNA, denatured DNA, and poly(U). It was found that poly(L -arginine) forms the α-helical conformation by interacting with the acidic homopolypeptides and the synthetic anionic polyelectrolytes. In each complex, poly(L -glutamic acid) is in the α-helical conformation, whereas poly(L -aspartic acid) is mostly in the random structure. The poly(L -glutamic acid) complex changed into the β-sheet structure at the transition temperature about 65°C in 0.01M cacodylate buffer (pH 7). Even in the presence of 5M urea, this complex remained in the α-helical conformation at room temperature. The existence of the stable complex of α-helical poly(L -arginine) and α-helical poly(L -glutamic acid) was successfully supported by the model building study of the complex. The α-helix of poly(L -arginine) induced by binding with polyacrylate was the most stable of the poly(L -arginine)-polyanion complexes examined as evidenced by thermal and urea effects. The lower helical content of the polyethylenesulfonate-complexed poly(L -aginine) was explained in terms of the higher charge density of the polyanion. On the other hand, native DNA, denatured DNA, and poly(U) were not effective in stabilizing the helical structure of poly(L -arginine). This may be due to the rigidity of polyanions and to the steric hindrance of bases. Furthermore, the distinitive structual behavior of poly(L -arginine) and poly(L -lysine) regarding polyanion interaction has been noticed throughout the study.     

The effect of calcium on the thermotropic properties of bovine blood coagulation factors IX and X and their activation intermediates and products

The thermotropic properties of bovine blood coagulation Factors IX and X, as well as the activation intermediates and products of these proteins, have been investigated by differential scanning microcalorimetry in the presence and absence of Ca²⁺. Bovine Factor IX displays a single thermal-denaturation transition characterized by a temperature midpoint (T_M) of 54.5 ± 0.5 °C and a calorimetric enthalpy (ΔH_c) of 105 ± 15 kcal/mol, in the absence of Ca²⁺. In the presence of Ca²⁺ concentrations sufficient to saturate its sites on Factor IX, the T_m value is increased to 57.0 ± 0.5 °C and the ΔH_c is virtually unchanged. When the activation intermediate, Factor IXα, is similarly analyzed in the absence of Ca²⁺, a broad, diffuse thermogram was obtained which did not lend itself to calculation of thermodynamic parameters. In the presence of Ca²⁺, Factor IXα displayed thermograms characterized by a T_M of 51.0 ± 0.5 °C and a ΔH_c of 109 ± 10 kcal/mol. The activated product, Factor IXaα, in the absence of Ca²⁺ (the values in the presence of saturating Ca²⁺ are given in parentheses), undergoes thermal denaturation with a T_M of 54.5 ± 0.5 °C (57.0 ± 0.5 °C) and a ΔH_c of 158 ±10 kcal/mol (156 ± 10 kcal/mol). Similarly, the terminal-activation product, Factor IXaβ, displays a T_M of 51.5 ± 0.5 °C (54.0 ± 0.5 °C) and a ΔH_c of 85 ± 5 kcal/mol (126 ± 10 kcal/mol). Bovine blood coagulation Factor X has been analyzed in this same fashion, and shows very similar thermal properties to Factor IX. The thermal denaturation of Factor X is represented by a T_M of 54.0 ± 0.5 °C (55.0 ± 0.5 °C) and a ΔH_c of 102 ± 10 kcal/mol (118 ± 10 kcal/mol), whereas its activated form, Factor Xaβ, possesses a T_M of 55.0 ± 0.5 °C (55.0 ± 0.5 °C) and a ΔH_c of 92.0 ± 5 kcal/mol (136 ± 10 kcal/mol). These studies indicate that, for many of these proteins, Ca²⁺ induces a conformational alteration to a more thermally stable form, which also requires the absorption of greater amounts of heat for thermal denaturation.