Study on the binding of Arsenazo-TB to human serum albumin by Rayleigh light scattering technique and FT-IR

The interaction between Arsenazo-TB and human serum albumin (HSA) was studied by Rayleigh light scattering (RLS) technique and Fourier transformed IR (FT-IR). The binding parameters of Arsenazo-TB with HSA were studied at different temperature of 288, 298, 308, 318 K under the optimum conditions. It is indicated by the Scatchard plots that the binding constant K decreased from 5.03 x 10(7) to 7.13 x 10(6) and the maximum binding number N reduced from 53 to 36 with the increasing of the temperature. The binding process was exothermic, enthalpy driven and spontaneous, as indicated by the thermodynamic analyses, and the major part of the binding energy is hydrophobic interaction. The free energy change deltaG0, the enthalpy change deltaH0 and the entropy change deltaS0 of 288 K were calculated to be -42.46 kJ/mol, -49.17 kJ/mol and 318.15 J/mol K, respectively. The alterations of protein secondary structure in the presence of Arsenazo-TB in aqueous solution were quantitatively calculated from FT-IR spectroscopy with reductions of alpha-helix from 57% to 40% and with increases of beta-sheet from 36% to 39%, beta-turn from 7% to 21%.     

Thermodynamic and structural characterization of cis-trans isomerization of 12-(S)-hydroxy-(5Z, 8E, 10E)-heptadecatrienoic acid by high-performance liquid chromatography and gaschromatography-mass spectrometry.

It is shown that 12-(S)-hydroxy-(5Z, 8E, 10E)-heptadecatrienoic acid (5-cis-HHT)--a physiological metabolite of arachidonic acid--is acid-catalyzed converted into a less polar substance with its maximum UV-absorption at (1)max=232 nm and a molar absorptivity of about epsilon=26600 +/- 200 M(-1)cm(-1). Using a reversed-phase high-performance liquid chromatographic (HPLC) method this equilibrium reaction (K(c) = 1.78 +/- 0.05 at pH 1.10 and 298 K) could be thermodynamicly characterized as a pH dependent, exergonic and exothermic reaction according to kinetics of a first order reaction (at pH 1.10 and 298 K: delta(R)G(o) = -1.42 +/- 0.07 kJ mol(-1), delta(R)H(o) = -3.50 +/- 0.9 kJ mol(-1), delta(R)S(o) = 7.0 +/- 3.0 J mol(-1)*K, delta(R)H*f = 100.0 +/- 4.0 kJ mol(-1)). Kinetic data for several pH-values and temperatures are presented. These data and structural characterization by gaschromatography-mass spectrometry (GC/MS) lead to the conclusion that 5-cis-HHT is isomerized to 12-(S)-hydroxy-(5E, 8E, 10E)-heptadecatrienoic acid (5-trans-HHT).     

Thermodynamics of the conversion of aqueous L-aspartic acid to fumaric acid and ammonia

The thermodynamics of the conversion of aqueous L-aspartic acid to fumaric acid and ammonia have been investigated using both heat conduction microcalorimetry and high-pressure liquid chromatography. The reaction was carried out in aqueous phosphate buffer over the pH range 7.25-7.43, the temperature range 13-43 degrees C, and at ionic strengths varying from 0.066 to 0.366 mol kg(-1). The following values have been found for the conversion of aqueous L-aspartateH- to fumarate2- and NH4+ at 25 degrees C and at zero ionic strength: K = (1.48 +/- 0.10) x 10(-3), DeltaG degrees = 16.15 +/- 0.16 kJ mol(-1), DeltaH degrees = 24.5 +/- 1.0 kJ mol(-1), and DeltaC(p) degrees = -147 +/- 100 J mol(-1) K(-1). Calculations have also been performed which give values of the apparent equilibrium constant for the conversion of L-aspartic acid to fumaric acid and ammonia as a function of temperature, pH and ionic strength.     

Salt bridges in the hyperthermophilic protein Ssh10b are resilient to temperature increases

A double mutant cycle (DMC) approach was employed to estimate the effect of temperature on the contribution of two highly conserved salt bridges to protein stability in the hyperthermophilic protein Ssh10b. The coupling free energy were 2.4 +/- 0.4 kJ/mol at 298 K and 2.2 +/- 0.4 kJ/mol at 353 K for Glu-54/Arg-57, and 6.0 +/- 0.2 kJ/mol at 298 K and 5.9 +/- 0.6 kJ/mol at 353 K for Glu-36/Lys-68. The stability free energy of Ssh10b decrease greatly with increasing temperature, while the direct contribution of these two salt bridges to protein stability remain almost constant, providing evidence supporting the theoretical prediction that salt bridges are extremely resilient to temperature increases and thus are specially suited to improving protein stability at high temperatures. The reason for the difference in coupling free energy between salt bridges Glu-54/Arg-57 and Glu-36/Lys-68 is discussed. Comparing our results with published DMC data for the contribution of salt bridges to stability in other proteins, we found that the energy contribution of a salt bridge formed by two charged residues far apart in the primary sequence is higher than that of those formed between two very close ones. Implications of this finding are useful for engineering proteins with enhanced thermostability.     

The effect of driving force on intramolecular electron transfer in proteins. Studies on single-site mutated azurins.

An intramolecular electron-transfer process has previously been shown to take place between the Cys3--Cys26 radical-ion (RSSR-) produced pulse radiolytically and the Cu(II) ion in the blue single-copper protein, azurin [Farver, O. & Pecht, I. (1989) Proc. Natl Acad. Sci. USA 86, 6868-6972]. To further investigate the nature of this long-range electron transfer (LRET) proceeding within the protein matrix, we have now investigated it in two azurins where amino acids have been substituted by single-site mutation of the wild-type Pseudomonas aeruginosa azurin. In one mutated protein, a methionine residue (Met44) that is proximal to the copper coordination sphere has been replaced by a positively charged lysyl residue ([M44K]azurin), while in the second mutant, another residue neighbouring the Cu-coordination site (His35) has been replaced by a glutamine ([H35Q]azurin). Though both these substitutions are not in the microenvironment separating the electron donor and acceptor, they were expected to affect the LRET rate because of their effect on the redox potential of the copper site and thus on the driving force of the reaction, as well as on the reorganization energies of the copper site. The rate of intramolecular electron transfer from RSSR- to Cu(II) in the wild-type P. aeruginosa azurin (delta G degrees = -68.9 kJ/mol) has previously been determined to be 44 +/- 7 s-1 at 298 K, pH 7.0. The [M44K]azurin mutant (delta G degrees = -75.3 kJ/mol) was now found to react considerably faster (k = 134 +/- 12 s-1 at 298 K, pH 7.0) while the [H35Q]azurin mutant (delta G degrees = -65.4 kJ/mol) exhibits, within experimental error, the same specific rate (k = 52 +/- 11 s-1, 298 K, pH 7.0) as that of the wild-type azurin. From the temperature dependence of these LRET rates the following activation parameters were calculated: delta H++ = 37.9 +/- 1.3 kJ/mol and 47.2 +/- 0.7 kJ/mol and delta S++ = -86.5 +/- 5.8 J/mol.K and -46.4 +/- 4.4 J/mol.K for [H35Q]azurin and [M44K]azurin, respectively. Using the Marcus relation for intramolecular electron transfer and the above parameters we have determined the reorganization energy, lambda and electronic coupling factor, beta. The calculated values fit very well with a through-bond LRET mechanism.     

Kinetic and structural characterization of manganese(II)-loaded methionyl aminopeptidases

Manganese(II) activation of the methionyl aminopeptidases from Escherichia coli (EcMetAP-I) and the hyperthermophilic archaeon Pyrococcus furiosus (PfMetAP-II) was investigated. Maximum catalytic activity for both enzymes was obtained with 1 equiv of Mn(II), and the dissociation constants (K(d)) for the first metal binding site were found to be 6 +/- 0.5 and 1 +/- 0.5 microM for EcMetAP-I and PfMetAP-II, respectively. These K(d) values were verified by isothermal titration calorimetry (ITC) and found to be 3.0 +/- 0.2 and 1.4 +/- 0.2 microM for EcMetAP-I and PfMetAP-II, respectively. The hydrolysis of MGMM was measured in triplicate between 25 and 85 degrees C at eight substrate concentrations ranging from 2 to 20 mM for PfMetAP-II. Both specific activity and K(m) values increased with increasing temperature. An Arrhenius plot was constructed from the kcat values and was found to be linear over the temperature range 25-85 degrees C. The activation energy for the Mn(II)-loaded PfMetAP-II hydrolysis of MGMM was found to be 25.7 kJ/mol while the remaining thermodynamic parameters calculated at 25 degrees C are DeltaG+ = 50.1 kJ/mol, DeltaH+ = 23.2 kJ/mol, and DeltaS++ = -90.2 J x mol(-1) x K(-1).     

pH-dependent redox activity and fluxionality of the copper site in amicyanin from Thiobacillus yersutus as studied by 300- and 600- MHz 1H NMR

The kinetics of the deuteronation of one of the copper ligand histidines of the reduced Type I blue-copper protein amicyanin from Thiobacillus versutus was studied as a function of temperature by 300- and 600- MHz 1H NMR. The NMR data were analyzed with the help of a three site exchange model. Deuteron exchange between the histidine ligand and the solution appears to be catalyzed by phosphate. After deuteronation the histidine can occur in two conformations. The electron self-exchange rate of amicyanin was determined as a function of temperature and ionic strength. At 298 K, pD = 8.6, I = 0.05 M, the ese rate amounts to 1.3 x 10(5) M-1 S-1. The activation parameters amount to delta H not equal to = (52 +/- 3) kJ/mol and delta S not equal to = (26 +/- 9) J/mol.K. The dependence of the ese rate on ionic strength is small. The deuteronated amicyanin appears to be redox-inactive. The experimental findings clearly distinguish amicyanin from other classes of blue-copper proteins like the azurins and the pseudo-azurins.     

Conformational properties of streptokinase--differential scanning calorimetric investigations.

The two-domain structure of streptokinase (Sk) was demonstrated by scanning calorimetric investigations at neutral pH and low ionic strength. The melting pattern of the protein is composed of two two-state transitions at TtrS1 = 45.9 +/- 0.4 degrees C with delta H1 = 431 +/- 18 kJ/mol, and TtrS2 = 60.1 +/- 1.3 degrees C with delta H2 = 306 +/- 16 kJ/mol. The partial specific heat capacity of native Sk was determined to be Cp = 1.42 +/- 0.17 J/K/g and the denaturational heat capacity change associated with the two transitions, delta Cp1 = 0.21 J/K/g and delta Cp2 = 0.38 J/K/g, respectively. The overall melting pattern of Sk remains almost unchanged at a variety of tested solvent compositions, except at pH 4 (and below) and in the presence of denaturants. The two domains show different susceptibility to urea. It is proposed that the less thermostable domain is located within the N-terminal part (residues 1-230), and the more thermostable one, within the C-terminal region.     

Thermodynamics of active-site ligand binding to Escherichia coli glutamine synthetase

Active-site ligand interactions with dodecameric glutamine synthetase from Escherichia coli have been studied by calorimetry and fluorometry using the nonhydrolyzable ATP analogue 5'-adenylyl imidodiphosphate (AMP-PNP), L-glutamate, L-Met-(S)-sulfoximine, and the transition-state analogue L-Met-(S)-sulfoximine phosphate. Measurements were made with the unadenylylated enzyme at pH 7.1 in the presence of 100 mM KCl and 1.0 mM MnCl2, under which conditions the two catalytically essential metal ion sites per subunit are occupied and the stoichiometry of active-site ligand binding is equal to 1.0 equiv/subunit. Thermodynamic linkage functions indicate that there is strong synergism between the binding of AMP-PNP and L-Met-(S)-sulfoximine (delta delta G' = -6.4 kJ/mol). In contrast, there is a small antagonistic effect between the binding of AMP-PNP and L-glutamate (delta delta G' = +1.4 kJ/mol). Proton effects were negligible (less than or equal to 0.2 equiv of H+ release or uptake/mol) for the different binding reactions. The binding of AMP-PNP (or ATP) to the enzyme is entropically controlled at 303 K with delta H = +5.4 kJ/mol and delta S = +150 J/(K.mol). At 303 K, the binding of L-glutamate (delta H = -22.2 kJ/mol) or L-Met-(S)-sulfoximine [delta H = -45.6 kJ/mol with delta Cp approximately equal to -670 +/- 420 J/(K.mol)] to the AMP-PNP.Mn.enzyme complex is enthalpically controlled with opposing delta S values of -29 or -46 J/(K.mol), respectively. The overall enthalpy change is negative and the overall entropy change is positive for the simultaneous binding of AMP-PNP and L-glutamate or of AMP-PNP and L-Met-(S)-sulfoximine to the enzyme. For the binding of the transition-state analogue L-Met-(S)-sulfoximine phosphate (which inactivates the enzyme by blocking active sites), both enthalpic and entropic contributions also are favorable at 303 K [delta G' approximately equal to -109 and delta H = -54.8 kJ/mol of subunit and delta S approximately equal to +180 J/(K.mol)].     

Temperature dependence of the backbone dynamics of ribonuclease A in the ground state and bound to the inhibitor 5'-phosphothymidine (3'-5')pyrophosphate adenosine 3'-phosphate

The interaction of the dinucleotide inhibitor 5'-phosphothymidine(3',5')pyrophosphate adenosine 3'-phosphate (pTppAp) with bovine pancreatic ribonuclease A (RNase A) was characterized by calorimetry and solution NMR spectroscopy. Calorimetric data show that binding of pTppAp to RNase A is exothermic (DeltaH = -60.1 +/- 4.1 kJ/mol) with a dissociation constant of 16 nM at 298 K. At this temperature, the binding results in an entropy loss (TDeltaS = -16.8 +/- 7.3 kJ/mol) that is more favorable than that with the product analogue, 2'-CMP (TDeltaS = -31.3 +/- 0.9 kJ/mol). Temperature-dependent calorimetric experiments give a DeltaC(p) for ligand binding of -230 +/- 100 J/mol K. Binding of pTppAp results in noticeable effects on the backbone amide chemical shifts and dynamics. Amide backbone (15)N NMR spin-relaxation studies were performed on both apo RNase A and RNase A/pTppAp as a function of temperature. At each temperature, the model-free-determined order parameters, S(2), were significantly higher for RNase A/pTppAp than for the apo enzyme indicating a decrease in the conformational entropy of the protein upon ligand binding. Furthermore, the magnitude of this difference varies along the amino acid sequence specifically locating the entropic changes. The temperature dependence of S(2) at each residue enabled assessment of the local heat capacity changes (DeltaC(p)) from ligand binding. In an overall, average sense, DeltaC(p) for the protein backbone, determined from the NMR dynamics measurements, did not differ between apo RNase A and RNase A/pTppAp indicating that backbone dynamics contribute little to DeltaC(p) for protein-ligand interactions in this system. However, residue-by-residue comparison of the temperature-dependent change in entropy (DeltaS(B)) between free and bound forms reveals nonzero contributions to DeltaC(p) at individual sites. The balance of positive and negative changes reveals a redistribution of energetics upon binding. Furthermore, experiment and semiempirical estimates suggest that a large negative DeltaC(p) should accompany binding of pTppAp, and we conclude that this contribution must arise from factors other than amide backbone dynamics.     

A calorimetric and equilibrium investigation of the hydrolysis of lactose

The thermodynamics of the hydrolysis of lactose to glucose and galactose have been investigated using both high pressure liquid chromatography and heat-conduction microcalorimetry. The reaction was carried out over the temperature range 282-316 K and in 0.1 M sodium acetate buffer at a pH of 5.65 using the enzyme beta-galactosidase to catalyze the reaction. For the process lactose(aq) + H2O(liq) = glucose(aq) + galactose(aq), delta G0 = -8.72 +/- 0.20 kJ.mol-1, K0 = 34 +/- 3, delta H0 = 0.44 +/- 0.11 kJ.mol-1, delta S0 = 30.7 +/- 0.8 J.mol-1.K-1, and delta Cop = 9 +/- 20 J.mol-1.K-1 at 298.15 K. The standard state is the hypothetical ideal solution of unit molality. Thermochemical cycle calculations using enthalpies of combustion and solution, entropies, solubilities, activity coefficients, and apparent molar heat capacities have also been performed. These calculations indicate large discrepancies which are attributable primarily to errors in literature data on the enthalpies of combustion and/or third law entropies of the crystalline forms of the substrates.     

Positive heat capacity change upon specific binding of translation initiation factor eIF4E to mRNA 5' cap

Specific recognition of the mRNA 5' cap by eukaryotic initiation factor eIF4E is a rate-limiting step in the translation initiation. Fluorescence spectroscopy and high-sensitivity isothermal titration calorimetry were used to examine the thermodynamics of eIF4E binding to a cap-analogue, 7-methylGpppG. A van't Hoff plot revealed nonlinearity characterized by an unexpected, large positive molar heat capacity change (DeltaC(degree)(p) = +1.92 +/- 0.93 kJ.mol(-1).K(-1)), which was confirmed by direct ITC measurements (DeltaC(degree)(p) = +1.941 +/- 0.059 kJ.mol(-1).K(-1)). This unique result appears to come from an extensive additional hydration upon binding and charge-related interactions within the binding site. As a consequence of the positive DeltaC(degree)(p), the nature of the thermodynamic driving force changes with increasing temperature, from enthalpy-driven and entropy-opposed, through enthalpy- and entropy-driven in the range of biological temperatures, into entropy-driven and enthalpy-opposed. Comparison of the van't Hoff and calorimetric enthalpy values provided proof for the ligand protonation at N(1) upon binding, which is required for tight stabilization of the cap-eIF4E complex. Intramolecular self-stacking of the dinucleotide cap-analogue was analyzed to reveal the influence of this coupled process on the thermodynamic parameters of the eIF4E-mRNA 5' cap interaction. The temperature-dependent change in the conformation of 7-methylGpppG shifts significantly the intrinsic DeltaH(degree)(0) = -72.9 +/- 4.2 kJ.mol(-1) and DeltaS(degree)(0) = -116 +/- 58 J.mol(-1).K(-1) of binding to the less negative resultant values, by DeltaH(degree)(sst) = +9.76 +/- 1.15 kJ.mol(-1) and DeltaS(degree)(sst) = +24.8 +/- 2.1 J.mol(-1).K(-1) (at 293 K), while the corresponding DeltaC(degree)(p)(sst) = -0.0743 +/- 0.0083 kJ.mol(-1).K(-1) is negligible in comparison with the total DeltaC(degree)(p) .     

Specific DNA binding by the homeodomain Nkx2.5(C56S): detection of impaired DNA or unfolded protein by isothermal titration calorimetry

Titrations of specific 18-bp duplex DNA with the cardiac-specific homeodomain Nkx2.5(C56S) have utilized an ultrasensitive isothermal titration calorimeter (ITC). As the free DNA nears depletion, we observe large apparent decreases in the binding enthalpy when the DNA is impaired or when the temperature is sufficiently high to produce some unfolding of the free protein. Either effect can be attributed to refolding of the biopolymer that occurs as a result of stabilization due to the large favorable change in free energy on the homeodomain binding to DNA (-49.4 kJ/mol at 298 K). In either case, thermodynamic parameters obtained in such ITC experiments are unreliable. By using a lower temperature (85 vs. 95 degrees C) during the annealing of complementary DNA strands, damage of the 18-bp duplex DNA (T(m) = 72 degrees C) is avoided, and titrations with the homeodomain are normal at temperatures from 10 to 40 degrees C when >95% of the protein is folded. Under the latter conditions, the heat capacity plot is linear with a DeltaC(p) value of -0.80 +/- 0.03 kJ K(-1) mol(-1), which is more negative than that calculated from the burial of solvent accessible surface areas (-0.64 +/- 0.05 kJ K(-1) mol(-1)), consistent with water structures being at the protein-DNA interfaces.     

Complementary addressed modification of a hairpin-shaped model oligodeoxyribonucleotide

A hairpin-shaped oligodeoxyribonucleotide d(pTTGGCACGAGCAGCCAA) (I) was alkylated with the reagent d(TTGGG) greater than UCHRCl (RCl = -C6H5-N(CH3)-CH2-CH2Cl) complementary to the hairpin's stem. Thermodynamic parameters for the hairpin structure estimated from melting curves were: delta Hh = -125 +/- 17 kJ/mol, delta Sh = -380 +/- 84 J/mol.K; and for the reagent - target complex delta Hpx = -155 +/- 8 kJ/mol, delta Spx = -427 +/- 21 J/mol.K. Effective constants of association Kx of the oligonucleotide with the reagent were determined at 30 and 50 degrees from the concentration dependence of the reaction yield and were 1988 +/- 83 and 1239 +/- 58 M-1, respectively. Experimental values of Kx agreed with the values of Kx = Kpx/(1 + Kh), calculated with the use of the thermodynamic parameters.     

Thermodynamic dependence of DNA/DNA and DNA/RNA hybridization reactions on temperature and ionic strength

The thermodynamics of 5'-ATGCTGATGC-3' binding to its complementary DNA and RNA strands was determined in sodium phosphate buffer under varying conditions of temperature and salt concentration from isothermal titration calorimetry (ITC). The Gibbs free energy change, DeltaG degrees of the DNA hybridization reactions increased by about 6 kJ mol(-1) from 20 degrees C to 37 degrees C and exhibited heat capacity changes of -1.42 +/- 0.09 kJ mol(-1) K(-1) for DNA/DNA and -0.87 +/- 0.05 kJ mol(-1) K(-1) for DNA/RNA. Values of DeltaG degrees decreased non-linearly by 3.5 kJ mol(-1) at 25 degrees C and 6.0 kJ mol(-1) at 37 degrees C with increase in the log of the sodium chloride concentration from 0.10 M to 1.0 M. A near-linear relationship was observed, however, between DeltaG degrees and the activity coefficient of the water component of the salt solutions. The thermodynamic parameters of the hybridization reaction along with the heat capacity changes were combined with thermodynamic contributions from the stacking to unstacking transitions of the single-stranded oligonucleotides from differential scanning calorimetry (DSC) measurements, resulting in good agreement with extrapolation of the free energy changes to 37 degrees C from the melting transition at 56 degrees C.     

Dynamics of interaction of vitamin C with some potent nitrovasodilators, S-nitroso-N-acetyl-d,l-penicillamine (SNAP) and S-nitrosocaptopril (SNOCap), in aqueous solution

The reductive decomposition of both SNAP and SNOCap by ascorbate in aqueous solution (in the presence of EDTA) was thoroughly investigated. Nitric oxide (NO) release from the reaction occurs in an ascorbate concentration and pH dependent manner. Rates and hence NO release increased drastically with increasing pH, signifying that the most highly ionized form of ascorbate is the more reactive species. The experiments were monitored spectrophotometrically, and second-order rate constants calculated at 37 degrees C for the reduction of SNAP are k(b)=9.81+/-1.39 x 10(-3) M(-1) s(-1) and k(c)=662+/-38 M(-1) s(-1) and for SNOCap are k(b)=2.57+/-1.29 x 10(-2) M(-1) s(-1) and k(c)=49.7+/-1.3 M(-1) s(-1). k(b) and k(c) are the second-order rate constants via the ascorbate monoanion (HA-) and dianion (A2-) pathways, respectively. Activation parameters were also calculated and are DeltaHb++ =93+/-7 kJ mol(-1), DeltaSb++ =15+/-2 J K(-1) mol(-1) and DeltaHc++ =51+/-5 kJ mol(-1), DeltaSc++ =-28+/-3 J K(-1) mol(-1) with respect to the reactions involving SNAP. Those for the reaction between SNOCap and ascorbate were calculated to be DeltaHb++ =63+/-11 kJ mol(-1), DeltaSb++ =-71+/-20 J K(-1) mol(-1) and DeltaHc++ =103+/-7 kJ mol(-1), DeltaSc++ =118+/-8 J K(-1) mol(-1). The effect of Cu2+/Cu+ ions on the reductive decompositions of these S-nitrosothiols was also investigated in absence of EDTA. SNOCap exhibits relatively high stability at near physiological conditions (37 degrees C and pH 7.55) even in the presence of micromolar concentrations of Cu2+, with decomposition rate constant being 0.011 M(-1) s(-1) in comparison to SNAP which is known to be more susceptible to catalytic decomposition by Cu2+ (second-order rate constant of 20 M(-1) s(-1) at pH 7.4 and 25 degrees C). It was also observed that the reductive decomposition of SNAP is not catalyzed by alkali metal ions, however, there was an increase in rate as the ionic strength increases from 0.2 to 0.5 mol dm(-3) NaCl.     

Calorimetric evidence for a native-like conformation of hen egg-white lysozyme dissolved in glycerol

Hen egg-white lysozyme, lyophilized from aqueous solutions of different pH (from pH 2.5 to 10.0) and then dissolved in water and in anhydrous glycerol, has been studied by high-sensitivity differential scanning microcalorimetry over the temperature range from 10 to 150 degrees C. All lysozyme samples exhibit a cooperative conformational transition in both solvents occurring between 10 and 100 degrees C. The transition temperatures in glycerol are similar to those in water at the corresponding pHs. The transition enthalpies in glycerol are substantially lower than in water but follow similar pH dependences. The transition heat capacity increment in glycerol does not depend on the pH and is 1.25+/-0.31 kJ mol(-1) K(-1), which is less than one fifth of that in water (6. 72+/-0.23 kJ mol(-1) K(-1)). The thermal transition in glycerol is reversible and equilibrium, as demonstrated for the pH 8.0 sample, and follows the classical two-state mechanism. In contrast to lysozyme in water, the protein dissolved in glycerol undergoes an additional, irreversible cooperative transition with a marginal endothermic heat effect at temperatures of 120-130 degrees C. The transition temperature of this second transition increases with the heating rate which is characteristic of kinetically controlled processes. Thermodynamic analysis of the calorimetric data reveals that the stability of the folded conformation of lysozyme in glycerol is similar to that in water at 20-80 degrees C but exceeds it at lower and higher temperatures. It is hypothesized that the thermal unfolding in glycerol follows the scheme: N ifho-MG-->U, where N is a native-like conformation, ho-MG is a highly ordered molten globule state, and U is the unfolded state of the protein.     

High-pressure effect on the equilibrium and kinetics of cyanide binding to chloroperoxidase

The kinetics of cyanide binding to chloroperoxidase were studied using a high-pressure stopped-flow technique at 25 degrees C and pH 4.7 in a pressure range from 1 to 1000 bar. The activation volume change for the association reaction is delta V not equal to + = -2.5 +/- 0.5 ml/mol. The total reaction volume change, determined from the pressure dependence of the equilibrium constant, is delta V degrees = -17.8 +/- 1.3 ml/mol. The effect of temperature was studied at 1 bar yielding delta H not equal to + = 29 +/- 1 kJ/mol, delta S not equal to + = -58 +/- 4 J/mol per K. Equilibrium studies give delta H degrees = -41 +/- 3 kJ/mol and delta S degrees = -59 +/- 10 J/mol per K. Possible contributions to the binding process are discussed: changes in spin state, bond formation and conformation changes in the protein. An activation volume analog of the Hammond postulate is considered.     

The enthalpy of acyl chain packing and the apparent water-accessible apolar surface area of phospholipids

The energetics of phospholipid aggregation depend on the apparent water-accessible apolar surface area (ASAap), ordering effects of the chains, and headgroup interactions. We quantify the enthalpy and entropy of these interactions separately. For that purpose, the thermodynamics of micelle formation of lysophosphatidylcholines (LPCs, chains C10, C12, C14, and C16) and diacylphosphatidylcholines (DAPCs, chains C5, C6) and C7) are studied using isothermal titration calorimetry. The critical micelle concentration (CMC) values are 90, 15, and 1.9 mM (C5-C7-DAPC) and 6.8, 0.71, 0.045, and 0.005 mM (LPCs). The group contributions per methylene of DeltaDeltaG(0) = -3.1 kJ/mol and DeltaDeltaC(P) = -57 J/(mol. K) for LPCs agree with literature data on hydrocarbons and amphiphiles. An apparent deviation of DAPCs (-2.5 kJ/mol, 45 J/(mol. K)) is due to an intramolecular interaction between the two chains, burying 20% of the surface. The chain/chain interaction enthalpies in a micelle core are by approximately -2 kJ/(mol) per methylene group more favorable than in bulk hydrocarbons. We conclude that the impact of the chain conformation and packing on the interaction enthalpy is very pronounced. It serves to explain a variety of effects reported on membrane binding. Interactions within the water-accessible region show considerable DeltaH, but almost no DeltaG(0). The heat capacity changes suggest about three methylene groups (ASAap approximately 100 A2) per LPC remain exposed to water in a micelle (DAPC: 2 CH2/70 A2).     

Thermodynamics of carbohydrate isomerization reactions. The conversion of aqueous allose to psicose

The thermodynamics of the conversion of aqueous D-psicose to D-allose has been investigated using high-pressure liquid chromatography. The reaction was carried out in phosphate buffer at pH 7.4 over the temperature range 317.25-349.25 K. The following results are obtained for the conversion process at 298.15 K: DeltaG degrees = - 1.41 +/- 0.09 kJ mol(-1), DeltaH degrees = 7.42 +/- 1.7 kJ mol(-1), and DeltaC(p) degrees = 67 +/- 50 J mol(-1) K(-1). An approximate equilibrium constant of 0.30 is obtained at 333.15 K for the conversion of aqueous D-psicose to D-altrose. Available thermodynamic data for isomerization reactions involving aldohexoses and aldopentoses are summarized.