Effect of temperature on viscosity properties of some alpha-amino acids in aqueous urea solutions

Viscosities for solutions of glycine, DL-alpha-alanine, DL-alpha-amino-n-butyric acid, DL-valine, DL-leucine and L-serine in 5 mol kg(-1) aqueous urea have been determined at 278.15, 288.15, 298.15 and 308.15 K. The viscosity B-coefficients for the amino acids in the aqueous urea solution have been calculated at different temperatures. The effect of temperature on the B-coefficients is discussed on the basis of the Feakins equation. The contribution of solute to the activation parameters (delta mu0*2, deltaH0*2, deltaS0*2) for viscous flow of the solution have been calculated, together with the Gibbs energy, enthalpy and entropy of transfer for the amino acids from the ground-state solvent to the hypothetical viscous transition state solvent. The contributions of the charged end group (NH3+, COO-) and CH2 groups of the amino acids to B-coefficient and delta mu0*2 have been also estimated using the linear correlations between B-coefficient or delta mu0*2 and the number of carbon atoms in the alkyl chains of the amino acids. All the activation parameters are discussed in terms of the solute-solvent interactions in the ground and transition states.     

Binding of the recombinant proteinase inhibitor eglin c from leech Hirudo medicinalis to human leukocyte elastase, bovine alpha-chymotrypsin and subtilisin Carlsberg: thermodynamic study

The effect of pH and temperature on the apparent association equilibrium constant (Ka) for the binding of the recombinant proteinase inhibitor eglin c from leech Hirudo medicinalis to human leukocyte elastase (EC 3.4.21.37), bovine alpha-chymotrypsin (EC 3.4.21.1) and subtilisin Carlsberg (EC 3.4.21.14) has been investigated. On lowering the pH from 9.5 to 4.5, values of Ka for eglin c binding to the serine proteinases considered decrease thus reflecting the acid-pK shift of the invariant histidyl catalytic residue (His57 in human leukocyte elastase and bovine alpha-chymotrypsin, and His64 in subtilisin Carlsberg) from congruent to 6.9, in the free enzymes, to congruent to 5.1, in the enzyme:inhibitor adducts. At pH 8.0, values of the apparent thermodynamic parameters for eglin c binding are: human leukocyte elastase - Ka = 1.0 x 10(10) M-1, delta G phi = -13.4 kcal/mol, delta H phi = +1.8 kcal/mol, and delta S phi = +52 entropy units; bovine alpha-chymotrypsin -Ka = 5.0 x 10(9) M-1, delta G phi = -13.0 kcal/mol, delta H phi = +2.0 kcal/mol, and delta S phi = +51 entropy units; and subtilisin Carlsberg - Ka = 6.6 x 10(9) M-1, delta G phi = -13.1 kcal/mol, delta H phi = +2.0 kcal/mol, and delta S phi = +51 entropy units (values of Ka, delta G phi and delta S phi were obtained at 21 degrees C; values of delta H phi were temperature independent over the range explored, i.e. between 10 degrees C and 40 degrees C; 1 kcal = 4184J).(ABSTRACT TRUNCATED AT 250 WORDS)     

Enzyme-catalyzed decomposition of dibenzoyl peroxide in organic solvents

Catalytic activity of catalase (CAT, EC 1.11.1.6), immobilized on carbon black NORIT and soot PM-100, with respect to decomposition of dibenzoyl peroxide (BPO) in non-aqueous media (acetonitrile and tetrachloromethane), was investigated with a quantitative UV-spectrophotometrical approach. Progress of the above reaction was controlled by selected kinetic parameters: the apparent Michaelis constant (Km(app)), the specific rate constant (k(sp)), the activation energy (Ea), the maximum reaction rate (Vmax), and the Arrhenius' pre-exponential factor (Z0). Conclusions on the tentative mechanism of the catalytic process observed were drawn from the calculated values of the Gibbs energy of activation (deltaG*), the enthalpy of activation (deltaH*), and entropy of activation (deltaS*).     

Dynamics of carbon monoxide recombination to fully reduced cytochrome c oxidase in plant mitochondria after low-temperature flash photolysis.

Rebinding of CO to reduced cytochrome c oxidase in plant mitochondria has been monitored optically at 590-630 nm after flash photolysis at low temperature from 160 to 200 K. (1) Under 100%-CO saturation, CO rebinding exhibits a four-step mechanism. The thermodynamic parameters of the first phase have been determined; its activation energy, Ea1, is 38.9 kJ.mol-1 and its enthalpy, delta H+/-1, and entropy, delta S+/-1, of activation are respectively 37.5 kJ.mol-1 and -75.8J.mol-1.K-1. (2) When the CO concentration is decreased to 0.2%, rebinding still occurs according to a four-step mechanism. The rate constant of the first phase is CO-concentration-independent. Under non-saturating conditions there is only one CO molecule per occupied site. The rebinding mechanism does not require additional CO molecules to be present in the haem pocket. (3) Dual-wavelength scanning experiments failed to detect optical forms correlated with the resolved phases. (4) Results are discussed with respect to previous work related to CO rebinding to mammalian cytochrome c oxidase and myoglobin.     

Thermodynamic properties of ligand binding by monoclonal anti-fluorescyl antibodies

The effects of temperature on the binding of fluorescein by three monoclonal anti-fluorescyl antibodies (4-4-20, 20-19-1, and 20-20-3) were assessed by measurements of affinity constants (Ka) over a temperature range of 2-70 degrees C. Values for Ka were determined from the degree of ligand association by using fluorescence methodology. Curvilinear van't Hoff plots (ln Ka vs. T-1) were observed for all three antibodies, indicating that their standard enthalpy changes (delta Ho) were temperature dependent. This phenomenon was further investigated by plotting the changes in unitary free energy (delta Gu), standard enthalpy (delta Ho), and unitary entropy (delta Su) vs. temperature. Strong temperature dependencies were observed for enthalpy and entropy values, while free energy plots were only weakly dependent on temperature. At low temperatures (4 degrees C), entropy played a major role in the binding of fluorescein by all three antibodies, while enthalpy dominated at higher temperatures. This was a consequence of the negative heat capacity changes (delta Cpo approximately equal to -320 cal K-1 mol-1) observed for these antibodies, which produced a negative trend in both enthalpy and entropy values with increasing temperature. The negative heat capacity values also indicated that the hydrophobic effect was instrumental in the binding of fluorescein. Entropy changes were lower than expected for hydrophobic binding alone, suggesting that other forces were acting to mitigate the hydrophobic effect. One possibility was that the binding of fluorescein acted to restrain vibrational fluctuations in the active-site region, producing negative changes in both heat capacity and entropy.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding of the bovine and porcine pancreatic secretory trypsin inhibitor (Kazal) to human leukocyte elastase: a thermodynamic study.

The effect of pH and temperature on the apparent association equilibrium constant (Ka) for the binding of the bovine and porcine pancreatic secretory trypsin inhibitor (Kazal-type inhibitor, PSTI) to human leukocyte elastase has been investigated. At pH 8.0, values of the apparent thermodynamic parameters for human leukocyte elastase: Kazal-type inhibitor complex formation are: bovine PSTI--Ka = 6.3 x 10(4) M-1, delta G degree = -26.9 kJ/mol, delta H degree = +11.7 kJ/mol, and delta S degree = +1.3 x 10(2) entropy units; porcine PSTI--Ka = 7.0 x 10(3) M-1, delta G degree = -21.5 kJ/mol, delta H degree = +13.0 kJ/mol, and delta S degree = +1.2 x 10(2) entropy units (values of Ka, delta G degree and delta S degree were obtained at 21.0 degrees C; values of delta H degree were temperature independent over the range (between 5.0 degrees C and 45.0 degrees C) explored). On increasing the pH from 4.5 to 9.5, values of Ka for bovine and porcine PSTI binding to human leukocyte elastase increase thus reflecting the acidic pK-shift of the His57 catalytic residue from congruent to 7.0, in the free enzyme, to congruent to 5.1, in the serine proteinase: inhibitor complexes. Thermodynamics of bovine and porcine PSTI binding to human leukocyte elastase has been analyzed in parallel with that of related serine (pro)enzyme/Kazal-type inhibitor systems. Considering the known molecular models, the observed binding behaviour of bovine and porcine PSTI to human leukocyte elastase was related to the inferred stereochemistry of the serine proteinase/inhibitor contact region(s).     

Effect of immobilization on kinetic and thermodynamic characteristics of sulfide oxidase from Arthrobacter species

In order to determine the impact of immobilization on biocatalytic efficacy of sulfide oxidase, the kinetic and thermodynamic properties of native and DEAE-cellulose immobilized sulfide oxidase from Arthrobacter species FR-3 were evaluated. Immobilization increased the catalytic efficiency of sulfide oxidase by producing a lower Michaelis-Menten constant (Km) and a higher rate of catalysis (Vmax) at different temperatures. The first-order kinetic analysis of thermal denaturation demonstrated that the values of enthalpy (delta H*d) and entropy (delta S*d) of immobilized sulfide oxidase were lower than the native enzyme, confirming the thermal stabilization of sulfide oxidase by immobilization. The delta H*d and delta S*d of the immobilized enzyme at 35 degrees C were 138.07 kJ/mol and 122.04 J/K/mol, respectively. These results suggest that immobilization made the sulfide oxidase from Arthrobacter sp. FR-3 thermodynamically more efficient for catalysis of sulfide oxidation.     

Kinetics and mechanism of electron transfer between meso-tetra sulphonated phenyl porphyrin iron(III)-apomyoglobin and Fe(EDTA)2- complexes

The kinetics of electron transfer between Fe(EDTA)2- and meso-tetra sulphonated phenyl porphyrin iron(III)-apomyoglobin have been studied by applying stopped-flow mixing and monitoring photometric changes at soret band (429 nm). The studies were carried out at pH's 6, 6.5, 7, 7.5, and 8 and at temperature between 10 and 40 degrees C. The mechanism proposed on the basis of the dependence of kobsd on Fe(EDTA)2- concentrations at various pH's, followed the rate equation: kobsd = ka[H+] + Kakb/[H+] + Ka.[Fe(EDTA)2-] The values of rate parameters calculated using a weighted non-linear least-squares analysis were: ka, 528 +/- 2 sec-1; kb, 25 +/- 1 sec-1; and Ka, 2.0 +/- 0.1 microM at 25 degrees C and 0.5 M sodium phosphate, and those of thermodynamic parameters calculated by the Eyring equation were: delta H*, 8.1 +/- 0.3 kcal mole-1 and delta S*, -23.4 +/- 1.1 eu at pH 7 and 0.5 M sodium phosphate.     

Thermodynamics of the Ca2+ binding to bovine alpha-lactalbumin

Bovine alpha-lactalbumin contains one strong Ca2+-binding site. The free energy (delta G0), enthalpy (delta H0), and entropy (delta S0) of binding of Ca2+ to this site have been calculated from microcalorimetric experiments. The enthalpy of binding was dependent on the metal-free bovine alpha-lactalbumin concentration. At 0.8 mg ml-1, metal-free bovine alpha-lactalbumin delta H0 was -110 +/- 6 kJ mol-1. At this concentration the binding constant was estimated from a mathematical analysis of the titration curves to be greater than 10(7) M-1. This means that delta G0 is smaller than -40 kJ mol-1 and delta S0 is less negative than -235 J.K-1 mol-1. The binding of Ca2+ is therefore enthalpy-driven. From binding experiments as a function of temperature, a delta Cp value of -4.1 kJ.K-1 mol-1 was calculated. This value is dependent on the protein concentration. A tentative explanation for this large value is given.     

Temperature dependence of the kinetics of the urea-induced dissociation of human plasma alpha 2-macroglobulin into half-molecules. A minimum rate at 15 degrees C indicates hydrophobic interaction between the subunits.

The kinetics of the urea-induced dissociation of human plasma alpha 2-macroglobulin to half-molecules has been studied as a function of temperature by using small-angle scattering of X-rays and neutrons. The most striking result of the present investigation is that there is a minimum in reaction rate at about 15 degrees C, and that the rate increases when the temperature is lowered, or raised, from that value. By analyzing the first-order rate constants in terms of transition-state theory it was found that the dissociation is associated with a large and positive change in heat capacity between the activated complex and native alpha 2-macroglobulin (delta CP is in the range 5 to 6 kJ mol-1 K-1). In analogy with pure thermodynamic investigations, where a large change in heat capacity normally is interpreted as a melting of hydrophobic interaction, we therefore propose that hydrophobic interaction is involved in the so-called non-covalent interactions between the subunits of alpha 2-macroglobulin. As a result of the present investigation, it also follows that the free energy of activation delta G has a maximum at about 32 degrees C, whereas the enthalpy of activation delta H and the entropy of activation delta S are zero at about 15 degrees C and 32 degrees C, respectively. These temperatures are slightly dependent upon the concentration of urea and upon whether the reaction is run in a 1H or a 2H medium. Furthermore, from the kinetic point of view, at low temperature the reaction can be characterized as enthalpy driven, whereas at high temperature, it can be characterized as entropy driven.     

Activation parameters for the carbonic anhydrase II-catalyzed hydration of CO2

We have determined the activation parameters of kcat and kcat/Km for the carbonic anhydrase II-catalyzed hydration of CO2. The enthalpy and entropy of activation for kcat is 7860 +/- 120 cal mol-1 and -3.99 +/- 0.42 cal mol-1 K-1, respectively, for the human enzyme. Results for the bovine enzyme were statistically indistinguishable from those of the human enzyme. The entropy of activation of kcat for the human enzyme was further decomposed into partially compensating electrostatic(es) (delta S*es = +15.1 cal mol-1 K-1) and nonelectrostatic(nes) (delta S*nes = -19.1 cal mol-1 K-1) terms. Computer simulations of a formal kinetic mechanism for carbonic anhydrase II-catalyzed CO2 hydration show that 82% of the temperature effect on kcat can be attributed to the temperature effect on the intramolecular proton transfer step. The reported activation parameters are consistent with a substantial enzyme or active site solvent conformational change in the transition state of the intramolecular proton transfer step, and is consistent with the mechanism of proton transfer proposed by Venkatasubban and Silverman (Venkatasubban, K. S., and Silverman, D. N. (1980) Biochemistry 19, 4984-4989).     

Thermodynamics of reversible monomer-dimer association of tubulin

The equilibrium between the rat brain tubulin alpha beta dimer and the dissociated alpha and beta monomers has been studied by analytical ultracentrifugation with use of a new method employing short solution columns, allowing rapid equilibration and hence short runs, minimizing tubulin decay. Simultaneous analysis of the equilibrium concentration distributions of three different initial concentrations of tubulin provides clear evidence of a single equilibrium characterized by an association constant, Ka, of 4.9 X 10(6) M-1 (Kd = 2 X 10(-7) M) at 5 degrees, corresponding to a standard free energy change on association delta G degrees = -8.5 kcal mol-1. Colchicine and GDP both stabilize the dimer against dissociation, increasing the Ka values (at 4.5 degrees C) to 20 X 10(6) and 16 X 10(6) M-1, respectively. Temperature dependence of association was examined with multiple three-concentration runs at temperatures from 2 to 30 degrees C. The van't Hoff plot was linear, yielding positive values for the enthalpy and entropy changes on association, delta S degrees = 38.1 +/- 2.4 cal deg-1 mol-1 and delta H degrees = 2.1 +/- 0.7 kcal mol-1, and a small or zero value for the heat capacity change on association, delta C p degrees. The entropically driven association of tubulin monomers is discussed in terms of the suggested importance of hydrophobic interactions to the stability of the monomer association and is compared to the thermodynamics of dimer polymerization.     

Thermal stability of Penicillium adametzii glucose oxidase]

The thermal stability of glucose oxidase was studied at temperatures between 50 and 70 degrees C by kinetic and spectroscopic (circular dichroism) methods. The stability of glucose oxidase was shown to depend on the medium pH, protein concentration, and the presence of protectors in the solution. At low protein concentrations (< 15 micrograms/ml) and pH > 5.5, the rate constants kin (s-1) for thermal inactivation of glucose oxidase were high. Circular dichroic spectra suggested an essential role of beta structures in stabilizing the protein globule. At a concentration of 15 micrograms protein/ml, the activation energy Ea of thermal inactivation of glucose oxidase in aqueous solution was estimated at 79.1 kcal/mol. Other thermodynamic activation parameters estimated at 60 degrees C had the following values: delta H = 78.4 kcal/mol, delta G = 25.5 kcal/mol, and delta S = 161.9 entropy units. The thermal inactivation of glucose oxidase was inhibited by KCl, polyethylene glycols, and polyols. Among polyols, the best was sorbitol, which stabilized glucose oxidase without affecting its activity. Ethanol, phenol, and citrate exerted destabilizing effects.     

Conformation of adenosine 3',5'-monophosphate in solution as studied by the NMR-desert method. II. Self-association and temperature-dependent glycosidic isomerization at pH 7.

A study has been made of the association and the temperature-dependent conformation of adenosine 3',5'-monophosphate (cyclic AMP) in a neutral aqueous (2H2O) solution by means of proton magnetic resonance chemical shift and relaxation. The concentration and temperature-dependent chemical shifts of H(1'), H(2), and H(8), have enabled us to estimate the self-association constant, Ka = 1.1 +/- 0.3 M-1 at 25 degrees C and thermodynamic parameters delta H = -5.8 +/- 1.5 kcal/mol and delta S (25 degrees C) = -19.0 +/- 3 cal/mol per degree. The NMR-DESERT (Deuterium Substitution Effect on Relaxation Times) method has been utilized for the determination of the syn-anti conformational equilibrium in the monomeric state and for the determination of the mutual orientation of the two adenine rings in the dimeric state of cyclic AMP. The molecules were found to coexist with nearly equimolarity or syn-anti conformers and thermal activation of the molecules perturbs the syn-anti conformational equilibrium to comprise the syn form in preference at higher temperature. The glycosidic isomerization (from anti to syn) was found to be characterized both by a positive enthalpy change and by a positive entropy change. The cyclic AMP molecules prefer to take a 'trans-stacking' conformation in the dimeric state where the two molecules are arranged in such a way that the H(2) of one molecule is close to the H(8) of the other.     

Binding of the trypsin inhibitor from white mustard (Sinapis alba L.) seeds to bovine beta-trypsin: thermodynamic study

The effect of pH and temperature on the association equilibrium constant (Ka) for the binding of the trypsin inhibitor from white mustard (Sinapis alba L.) seeds (MTI) to bovine beta-trypsin (EC 3.4.21.4) has been investigated. On lowering the pH from 9 to 3, values of Ka for MTI binding to bovine beta-trypsin decrease thus reflecting the acid-pK and -midpoint shifts, upon inhibitor association, of two independent ionizable groups, and of a three-proton transition, respectively. At pH 8.0, values of thermodynamic parameters for MTI binding to bovine beta-trypsin are: Ka = 4.5 X 10(8)M-1, delta G0 = -11.6 kcal/mol, and delta S0 = +53 entropy units (all at 21 degrees C); and delta H0 = +4.1 kcal/mol (temperature independent between 5 degrees C and 45 degrees C). Binding properties of MTI to bovine beta-trypsin have been analyzed in parallel with those concerning macromolecular inhibitor association to serine (pro)enzymes.     

Thermodynamic analyses of triplex formation with homopurine oligonucleotide.

We analyzed the thermodynamics of purine motif triplex formation by isothermal titration calorimetry. The signs of calorimetric enthalpy change, delta Hcal, and entropy change, delta S, of the triplex formation were negative in the temperature range between 15 and 35 degrees C. Since an observed negative delta S was unfavorable for the triplex formation, the triplex formation was driven by a large negative delta Hcal. delta Hcal decreased with increasing temperature, yielding a negative heat capacity change, delta Cp, of approximately -1.2 kcal mol-1 K-1. We found that the binding constant, Ka, increased with increasing temperature, leading to an apparent positive van't Hoff enthalpy change, delta Hvh, which was in sharp contrast with the large negative delta Hcal. The analyses of the observed temperature dependence of Ka and delta Hcal and the negative delta Cp suggest that the purine motif triplex formation near room temperature is not a simple two-state binding process but exhibits multiple states, which was previously observed for the pyrimidine motif triplex formation near room temperature.     

Thermodynamic analysis of rat brain opioid mu-receptor-ligand interaction

Opioid mu-receptors are membrane bound receptors. The mechanism by which they transduce their biological effect into the inner compartment of the postsynaptic cell is still not fully understood. The present study was attempted to the measurement of changes of the thermodynamic parameters of the receptor--agonist/antagonist interaction. We have set up the binding assays of a mu-receptor agonist (3H-dihydromorphine) as well as an antagonist (3H-naloxone). The saturation isotherms of both ligands have been assayed at various temperatures and from the resulting KD values the standard changes of Gibbs energy, enthalpy and entropy have been calculated. While the binding of the mu-receptor agonist 3H-dihydromorphine appears to be entropy driven (delta S0 = 230 J mol-1 K-1) and endothermic (delta H0 = 19 kJ mol-1), the binding of the mu-receptor antagonist 3H-naloxone is apparently driven by a decrease of standard enthalpy (delta H0 = -27 kJ mol-1; i.e. the reaction is exothermic) and is also characterized by an increase of standard entropy (delta S0 = 76 J mol-1 K-1). The maximal number of 3H-naloxone binding sites has to be determined by incubation at 0-4 degrees C. The present data to not support the view that opioid mu-receptors transduce their biological signal through the adenylatecyclase system by a mechanism similar to beta-adrenergically stimulated adenylatecyclase.     

17O and 14N n.m.r. studies of the Co (II) interaction with cyclo(Ala*-Ala) in aqueous solution

The complexation of cyclo(Ala*-Ala) with the cobaltous ions in aqueous solution was investigated by 17O and 14N n.m.r. spectroscopy. The 17O and 14N transverse relaxation time (T2p) and chemical shift (delta omega a) of cyclo(Ala*-Ala) were measured as a function of the temperature at pH = 7.03 +/- 0.02, and pH = 6.45 +/- 0.02, and as a function of pH at room temperature. No effects of pH on the transverse relaxation time and chemical shift were observed. Complementary 17O studies of the solvent water molecules were also carried out. The hyperfine coupling constant and the entropy and enthalpy of activation for the exchange of cyclo(Ala*-Ala) and water molecules between the coordinated and noncoordinated states were determined by least-square fit of theoretical equation for the chemical shift delta omega a to experimental data. The hyperfine coupling constant of the peptide bound oxygen was determined to be (-1.6 +/- 0.1) X 10(5) Hz and the entropy and enthalpy (32.0 +/- 3.0) kJ/mol and (-12.0 +/- 1.0) e.u, respectively. Information obtained from 17O n.m.r. study allows some inferences concerning the probable coordination sphere of the cobaltous ion. There are three types of complexes: Co(H2O)6(2+), CoL X 5H2O and CoL2 X 4H2O, with relative concentrations 19.9%, 2.9%, and 77.2%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermodynamics of the interaction of globular proteins with powdered stearic acid in acid pH

Adsorption isotherms of different globular proteins and gelatin on strearic acid particles have been studied as a function of biopolymer concentration, ionic strength of the medium, and temperature. The effect of neutral salts including CaCl2, Na3PO4, and urea on the adsorption isotherms has been also investigated. It is observed that the extent of adsorption (Gamma2(1)) increases in two steps with the increase of biopolymer concentration (C2) in the bulk. Gamma2(1) increases with an increase of C2 until a steady maximum value Gamma2(m) is reached at a critical concentration C2(m). After initial saturation, Gamma2(1) again increases from Gamma2(m) without reaching any limiting value due to the surface aggregation of the protein. The values of the standard free energy change for adsorption have been calculated on the basis of the Gibbs equation. The standard entropy and enthalpy changes are also calculated.     

The thermodynamic parameters of the conformational transitions in Mycoplasma DNA

DNA preparations have been isolated from 10 strains of phytopathogenic mycoplasms and collection culture Achole plasma laidlawii PG-8. Thermodynamic parameters of denaturation changes in the secondary structure (transconformation) of DNA of these mycoplasms have been determined. It is shown that denaturation temperature is 82.3-83.1 degree C and enthalpy of conformational DNA transitions calculated per 1 g of dry substance is 56.2-61.9 J/g. Changes in the enthalpy (delta H) and entropy (delta S) calculated per 1 mol of nucleotide pairs varied in the range of 35.6-39.0 J/m.p. and 995-109.6 J degree m.p. respectively. No linear dependence of transconformational thermal adsorption on the temperature of DNA denaturation of the studied strains of mycoplasms are revealed, which is probably connected with structural peculiarities of DNA of these microorganisms.