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.     

Thermodynamic studies of the reversible association of Escherichia coli ribosomal subunits.

The kinetics of association of Escherichia coli 30S and 50S ribosomal subunits have been carried out as a function of temperature after a magnesium jump from 1.5 to 3 mM. Turbidimetric recordings combined with a stopped-flow apparatus were used to follow the kinetics. The data show that the rates of formation and dissociation of the 70S particles at 3 mM Mg2+ and +25 degrees C were, respectively: k2 = 10(5) M-1 s-1, k1 = 4,5 X 10(-3) s-1; lowering the temperature decreases the rate constants with activation energies equal to E2 = 7.5 kcal/mol, E1 = 26.5 kcal/mol and enhances the association equilibrium towards the 70S species with an enthalpy change (delta H degrees assoc = -19.9 kcal/mol) dominant over the entropy change (delta S degrees assoc = -33 cal/(deg mol)). These thermodynamic parameters were compared to those obtained from studies on the interactions of codon-anticodon in yeast phenylalanine transfer RNA as well as of ribooligonucleotides. The kinetic and thermodynamic data are shown to be consistent with 16S-23S RNA interaction.     

Kinetic and thermodynamic evidence of a molybdate interaction with glucocorticoid receptor in calf thymus

The effect of molybdate on the kinetic and thermodynamic properties of the dexamethasone-receptor interaction was studied in calf thymus cytosol. In the presence of molybdate both the equilibrium binding studies and the association and dissociation experiments reveal a significantly lower affinity of the receptor for [3]dexamethasone. At 0 degrees C the equilibrium dissociation constant increases from 0.8 nM to 1.8 nM, the association rate constant shifts from 1.5 X 10(8) M-1 h-1 to 0.2 X 10(8) M-1 h-1, whereas the rate of dissociation of the untransformed receptor increases from 0.04 h-1 to 1.1 h-1 in the molybdate-containing buffer. All these effects appear dependent on the concentration of molybdate but the dissociation of the transformed receptor (0.01 h-1) is unaffected. The enthalpy for the association, delta H not equal to, increases at least twofold whereas the entropy, both for the association (delta S not equal to = -25 to +104 J K-1 mol-1) and for the equilibrium (delta S degrees = -100 to +38 J K-1 mol-1), is markedly influenced by the presence of molybdate. Taken all together these data suggest that molybdate interacts with the receptor molecule turning it into a form that displays low affinity for steroid, in addition to the well-documented incapacity to transform itself. This fact leads us to think that both the binding and the transformation are the expression of conformational modifications involving molybdate-sensitive groups.     

Temperature dependence of kinetic interactions between progesterone and the uterine cytoplasmic receptor

The temperature dependence of the rates of dissociation and association for progesterone-receptor interactions was measured over the temperature range of 0–20°C. The dissociation process is biphasic indicating that either two forms of receptor are present or that the binding of progesterone to the receptor is a concatenated reaction.The enthalpy of activation for the dissociation of progesterone from the receptor is about 26–28 kcal/mol and the entropic energy of activation is about ?5 kcal/mol. The enthalpy of activation for the association of these molecules is about 3 kcal/mol and the entropic energy of activation is about 6 kcal/mol. These data are consistent with a model of progesterone binding to the receptor that includes hydrogen bonds between each of the two ketone groups and hydrogen donors on the receptor protein and involves van der Waals' interactions, due to the close proximity of the receptor binding site to a large fraction of the progesterone surface.     

Mechanism of biphasic action of mono- and bivalent salts on the binding of prolactin to the receptor in the rabbits mammary gland

The influence of NaCl, KCl, CaCl2, and MgCl2 on the binding of prolactin (PRL) to its receptor was investigated. The salts were dissolved in a metallic ion-free binding buffer and had biphasic effects on changes in the association rate constant (k+1) of PRL binding, depending on their concentrations: there was an increase in the k+1 at lower concentrations and a decrease at higher concentrations. The dissociation rate of bound PRL was unaffected. NaCl at any concentration did not change the binding capacity. Bivalent salts, at higher than 25 mM, increased the capacity about 1.6-fold as compared to the 0 mM control. By cross-linking the PRL-receptor complex, the band of a molecular weight (Mr) 34,500 receptor could always be detected on the autoradiogram. An Mr 78,000 receptor appeared only after incubation with bivalent salts. Data indicate that the binding of PRL to an Mr 78,000 receptor is directly regulated by bivalent cation.     

Temperature dependence of the dissociation rate constants for 8 S, 4 S, and meroreceptor forms of the estrogen receptor from rat uterus

Conversion of a steroid receptor complex from the 8 S to the 4S form results in new interactions between the steroid and the receptor and/or formation of new intra-protein bonds within the receptor molecule itself. These bonds must be broken before the steroid is released. In order to localize these newly formed interactions, the dissociation kinetics of meroreceptors derived from 4 S and 8 S (molybdate-stabilized) receptor complexes were examined. At temperatures between 6 and 30 degrees C, no differences in the rates of dissociation were observed for the meroreceptors derived from the two forms of estrogen receptor, whereas approximately a twofold difference in dissociation rates for 4 S intact receptor versus 8 S intact receptor was detected. These findings indicate that the new interactions accompanying this conversion are likely to occur in regions of the receptor molecule other than the C-terminal portion of the steroid-binding site. The thermodynamic parameters of the dissociation reaction for the intact 4 S, and 8 S, and meroreceptor forms, respectively were: delta H [symbol; see text] = 26.2 +/- 1.3, 19.7 +/- 1.7, and 23.2 +/- 1.0 kcal/mol; +T delta S [symbol; see text] = 9.4 +/- 1.2, 3.2 +/- 1.7 and 6.6 +/- 0.9 kcal/mol (at 25 degrees C); and delta G [symbol; see text] = 16.8 +/- 2.5, 16.5 +/- 3.4, and 16.7 +/- 1.9 kcal/mol. As is the case for other steroid receptors, an increase in the enthalpy of steroid-receptor interaction after this conversion reflects the stability of the 4 S estrogen receptor complex.     

Thermodynamics of ion binding to phosphatidic acid bilayers. Titration calorimetry of the heat of dissociation of DMPA.

The heat of dissociation of the second proton of 1,2-dimyristoylphosphatidic acid (DMPA) was studied as a function of temperature using titration calorimetry. The dissociation of the second proton of DMPA was induced by addition of NaOH. From the calorimetric titration experiment, the intrinsic pK0 for the dissociation reaction could be determined by applying the Gouy-Chapman theory. pK0 decreases with temperature from ca. 6.2 at 11 degrees C to 5.4 at 54 degrees C. From the total heat of reaction, the dissociation enthalpy, delta Hdiss, was determined by subtracting the heat of neutralization of water and the heat of dilution of NaOH. In the temperature range between 2 and 23 degrees C, delta Hdiss is endothermic with an average value of ca. 2.5 kcal.mol-1 and shows no clear-cut temperature dependence. In the temperature range between 23 and 52 degrees C, delta Hdiss calculated after subtraction of the heat of neutralization and dilution is not the true dissociation enthalpy but includes contributions from the phase transition enthalpy, delta Htrans, as the pH jump induces a transition from the gel to the liquid-crystalline phase. The delta Cp for the reaction enthalpy observed in this temperature range is positive. Above 53 degrees C, the pH jump induces again only the dissociation of the second proton, and the bilayers stay in the liquid-crystalline phase. In this temperature range, delta Hdiss seems to decrease with temperature. The thermodynamic data from titration calorimetry and differential scanning calorimetry as a function of pH can be combined to construct a complete enthalpy-temperature diagram of DMPA in its two ionization states.     

Thermodynamic parameters and shape of the mycobacterial polymethylpolysaccharide-fatty acid complex

Properties of the mycobacterial polymethylpolysaccharide-lipid complex have been investigated by fluorometric techniques. From the dissociation constant for the O-methyglucose polysaccharide-parinaric acid complex at 293 K, a Gibbs free energy (delta G degree) of -33.65 kJ/mol was obtained. The Kd decreased with increasing temperature, giving an enthalpy (delta H degree) of 15.4 kJ/mol. From these data, a molar entropy (delta S degree) of 167.4 J K-1 was obtained. Thus, the reaction is slightly endothermic, but the large positive entropy change leads to an overall negative free energy favoring complex formation. From fluorescence depolarization measurements, the methylglucose polysaccharide-parinaric acid complex appears to display isotropic rotation with a correlation time of 2.55 ns at 23 degrees C. This may be compared to a rotational correlation time of 6.17 ps for free parinaric acid in water at 23 degrees C calculated from the value determined in cyclohexanol at the same temperature, which demonstrates that the mobility of the fatty acid in the complex is restricted. Assuming the complex is spherical, it was calculated to have a diameter of 23-26 A, whereas a helical methyglucose polysaccharide molecule assembled from space-filling models has the dimensions of a cylinder of 18 X 24 A. The polysaccharide and fatty acid chain-length dependence of the interaction shows a discontinuity for helical polysaccharide segments shorter than 12 sugars and for fatty acids shorter than palmitate.     

Temperature dependence of the preferential interactions of ribonuclease A in aqueous co-solvent systems: thermodynamic analysis.

The temperature dependence of preferential solvent interactions with ribonuclease A in aqueous solutions of 30% sorbitol, 0.6 M MgCl2, and 0.6 M MgSO4 at low pH (1.5 and 2.0) and high pH (5.5) has been investigated. This protein was stabilized by all three co-solvents, more so at low pH than high pH (expect 0.6 M MgCl2 at pH 5.5). The preferential hydration of protein in all three co-solvents was high at temperatures below 30 degrees C and decreased with a further increase in temperature (for 0.6 M MgCl2 at pH 5.5, this was not significant), indicating a greater thermodynamic instability at low temperature than at high temperature. The preferential hydration of denatured protein (low pH, high temperature) was always greater than that of native protein (high pH, high temperature). In 30% sorbitol, the interaction passed to preferential binding at 45% for native ribonuclease A and at 55 degrees C for the denatured protein. Availability of the temperature dependence of the variation with sorbitol concentration of the chemical potential of the protein, (delta mu(2)/delta m3)T,p,m2, permitted calculation of the corresponding enthalpy and entropy parameters. Combination with available data on sorbitol concentration dependence of this interaction parameter gave (approximate) values of the transfer enthalpy, delta H2,tr, and transfer entropy delta S2,tr. Transfer of ribonuclease A from water into 30% sorbitol is characterized by positive values of the transfer free energy, transfer enthalpy, transfer entropy, and transfer heat capacity. On denaturation, the transfer enthalpy becomes more positive. This increment, however, is small relative to both the enthalpy of unfolding in water and to the transfer enthalpy of the native protein from water a 30% sorbitol solution.     

The energetics of HMG box interactions with DNA: thermodynamics of the DNA binding of the HMG box from mouse sox-5

The energetics of the Sox-5 HMG box interaction with DNA duplexes, containing the recognition sequence AACAAT, were studied by fluorescence spectroscopy, isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC). Fluorescence titration showed that the association constant of this HMG box with the duplexes is of the order 4x10(7) M(-1), increasing somewhat with temperature rise, i.e. the Gibbs energy is -40 kJ mol(-1) at 5 degrees C, decreasing to -48 kJ mol(-1) at 32 degrees C. ITC measurements of the enthalpy of association over this temperature range showed an endothermic effect below 17 degrees C and an exothermic effect above, suggesting a heat capacity change on binding of about -4 kJ K(-1) mol(-1), a value twice larger than expected from structural considerations. A straightforward interpretation of ITC data in heat capacity terms assumes, however, that the heat capacities of all participants in the association reaction do not change over the considered temperature range. Our previous studies showed that over the temperature range of the ITC experiments the HMG box of Sox-5 starts to unfold, absorbing heat and the heat capacities of the DNA duplexes also increase significantly. These heat capacity effects differ from that of the DNA/Sox-5 complex. Correcting the ITC measured binding enthalpies for the heat capacity changes of the components and complex yielded the net enthalpies which exhibit a temperature dependence of about -2 kJ K(-1) mol(-1), in good agreement with that predicted on the basis of dehydration of the protein-DNA interface. Using the derived heat capacity change and the enthalpy and Gibbs energy of association measured at 5 degrees C, the net enthalpy and entropy of association of the fully folded HMG box with the target DNA duplexes was determined over a broad temperature range. These functions were compared with those for other known cases of sequence specific DNA/protein association. It appears that the enthalpy and entropy of association of minor groove binding proteins are more positive than for proteins binding in the major groove. The observed thermodynamic characteristics of protein binding to the A+T-rich minor groove of DNA might result from dehydration of both polar and non-polar groups at the interface and release of counterions. The expected entropy of dehydration was calculated and found to be too large to be compensated by the negative entropy of reduction of translational/rotational freedom. This implies that DNA/HMG box association proceeds with significant decrease of conformational entropy, i.e. reduction in conformational mobility.     

Mechanism of lectin-cell interactions: thermodynamic and kinetic analysis of the binding of winged bean acidic lectin (WBA II) to human erythrocytes.

In order to identify the forces involved in the binding and to understand the mechanism involved, equilibrium and kinetic studies were performed on the binding of the winged bean acidic lectin to human erythrocytes. The magnitudes of delta S and delta H were positive and negative respectively, an observation differing markedly from the lectin-simple sugar interactions where delta S and delta H are generally negative. Analysis of the sign and magnitudes of these values indicate that ionic and hydrogen bonded interactions prevail over hydrophobic interactions resulting in net -ve delta H (-37.12 kJ.mol-1) and +ve delta S (14.4 J.mole-1 K-1 at 20 degrees C), thereby suggesting that this entropy driven reaction also reflects conformational changes in the lectin and/or the receptor. Presence of two kinds of receptors for WBA II on erythrocytes, as observed by equilibrium studies, is consistent with the biexponential dissociation rate constants (at 20 degrees C K1 = 1.67 x 10(-3) M-1 sec-1 and K2 = 11.1 x 10(-3) M-1 sec-1). These two rate constants differed by an order of magnitude accounting for the difference in the association constants of the two receptors of WBA II. However, the association process remains monoexponential suggesting no observable difference in the association rates of the lectin molecule with both the receptors, under the experimental conditions studied. The thermodynamic parameters calculated from kinetic data correlate well with those observed by equilibrium. A two-step binding mechanism is proposed based on the kinetic parameters for WBA II-receptor interaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermodynamics of agonist and antagonist binding to the alpha 1-adrenoceptor studied using [125I]BE 2254

The thermodynamics of binding of [125I]BE 2254 to the alpha 1-adrenoceptor in guinea pig brain membranes have been investigated at four different temperatures between 0 and 37 degrees C. The affinity and binding capacity of the radioligand did not vary with temperature. Thus, the change in enthalpy upon binding was close to zero whereas the change in entropy was large and positive (delta S degrees approximately 45 cal/mol-deg). In addition, [125I]BE 2254 has been used as a reporter ligand to probe the thermodynamics of the interaction of a variety of alpha-adrenoceptor agonists and antagonists with the alpha 1-adrenoceptor. Binding of all ligands was associated with large positive changes in entropy (delta S degrees between 18 and 48 cal/mol-deg) and little, or no, change in enthalpy, a finding that provides no convincing evidence for conformational rearrangement of alpha 1-adrenoceptors upon ligand binding.     

Redox properties of rubredoxin variants as a function of solvent composition and temperature: investigation of monopolar and dipolar interactions

The role of solvent composition and temperature on equilibrium electron transfer in seven rubredoxin variants [ Clostridium pasteurianum ( Cp), V8D, V8R, V8A, V44A Cp, Pyrococcus furiosus ( Pf), and A44V Pf] were investigated to examine the role of both monopolar and dipolar interactions. The reduction potentials of all variants decreased as the polarity of the solvent decreased. The enthalpy and entropy associated with electron transfer were determined from temperature-controlled voltammetric studies. The entropic contribution [delta( Tdelta S degrees )] to the change in the reduction potential was larger for charged variants (V8D and V8R), while the enthalpic contribution [delta(-delta H degrees )] was larger for the other mutants. The large entropy change observed for monopolar variants is likely due to solvent reorganization that occurs between oxidation states. Entropic-enthalpic compensation phenomena, an observation that most proteins have an entropic term [delta( Tdelta S degrees )] and enthalpic term [delta(-delta H degrees )] with opposite signs, was observed. A correlation of the size of the amino acid side chain with delta E degrees ', delta(-delta H degrees ), and delta( Tdelta S degrees ) is also discussed.     

Slow inhibition of almond beta-glucosidase by azasugars: determination of activation energies for slow binding

The thermodynamic and activation energies of the slow inhibition of almond beta-glucosidase with a series of azasugars were determined. The inhibitors studied were isofagomine ((3R,4R,5R)-3,4-dihydroxy-5-hydroxymethylpiperidine, 1), isogalactofagomine ((3R,4S,5R)-3,4-dihydroxy-5-hydroxymethylpiperidine, 2), (-)-1-azafagomine ((3R,4R,5R)-4,5-dihydroxy-3-hydroxymethylhexahydropyridazine, 3), 3-amino-3-deoxy-1-azafagomine (4) and 1-deoxynojirimycin (5). It was found that the binding of 1 to the enzyme has an activation enthalpy of 56.1 kJ/mol and an activation entropy of 25.8 J/molK. The dissociation of the enzyme-1 complex had an activation enthalpy of -2.5 kJ/mol and an activation entropy of -297 J/molK. It is suggested that the activation enthalpy of association is due to the breaking of bonds to water, while the large negative activation entropy of dissociation is due at least in part to the resolvation of the enzyme with water molecules. For the association of 1 DeltaH(0) is 58.6 kJ/mol and DeltaS(0) is 323.8 J/molK. Inhibitor 3 has an activation enthalpy of 39.3 kJ/mol and an activation entropy of -17.9 J/molK for binding to the enzyme, and an activation enthalpy of 40.8 kJ/mol and an activation entropy of -141.0 J/molK for dissociation of the enzyme-inhibitor complex. For the association of 3 DeltaH(0) is -1.5 kJ/mol and DeltaS(0) is 123.1 J/molK. Inhibitor 5 is not a slow inhibitor, but its DeltaH(0) and DeltaS(0) of association are -30 kJ/mol and -13.1 J/molK. The large difference in DeltaS(0) of association of the different inhibitors suggests that the anomeric nitrogen atom of inhibitors 1-4 is involved in an interaction that results in a large entropy increase.     

Effect of temperature on dynamic viscoelasticity during the clotting reaction of fibrin

The dynamic rigidity and loss moduli for fibrinogen-thrombin solution were determined during clotting in the temperature range between 15 and 45 degrees C. The rigidity of fibrin gel decreased with increasing clotting temperature, owing to the dissociation of cross-links. The rate constant of the dissociation of cross-links increased with increasing temperature. The rate constant of the cross-linking reaction increased and then decreased through a maximum with increasing temperature. It is explained by assuming that denaturation of fibrin occurs at high temperature. The irreversible denaturation becomes appreciable at high ionic strength. The activation energy and the enthalpy change for the cross-linking reaction of fibrin is about 35 and 15 kcal/mol, respectively. The enthalpy change for the reversible denaturation is about 46 kcal/mol.     

Kinetics of the interaction of dihydroalprenolol with beta-adrenergic receptors in rat cerebral cortex

Time and temperature dependence of the binding of 3H-dihydroalprenolol (3H-DHA) to beta-adrenergic receptors in rat cerebral cortex is described. The kinetic data obtained suggest that 3H-DHA binding proceeds through a two-step reaction scheme consisting of a bimolecular association step followed by an unimolecular internal conversion of the radioligand receptor complex (isomerisation). Equilibrium thermodynamic analysis provided evidence that the over-all binding process is associated with a small decrease in enthalpy and a substantial increase in entropy. Within the framework of the two-step binding kinetics, the evaluation of the temperature dependence by the van't Hoff analysis resulted in values for thermodynamic parameters for the single equilibrium steps. The data suggest that the association step can be considered as a bimolecular hydrophobic interaction which is mainly entropy-driven due to the release of structural water, while the isomerisation step is accompanied by a large negative change in both enthalpy and entropy. The large negative change in the activation entropy for the forward reaction of the isomerisation step, obtained from evaluation of Arrhenius plots, indicates an internal conversion to a highly ordered receptor-ligand complex, while the low activation energy points to a small threshold energy for reaching this structure. Thus, these result support a previous assumption that the hydrophobic center of an adrenergic antagonist interacts with the receptor by entering a pocket (Cherksey et al. 1981).     

Dipole-dipole interaction stabilizes the transition state of apurinic/apyrimidinic endonuclease--abasic site interaction

Human apurinic/apyrimidinic (AP) endonuclease (hAPE) initiates the repair of an abasic site (AP site). To gain insight into the mechanisms of damage recognition of hAPE, we conducted surface plasmon resonance spectroscopy to study the thermodynamics and kinetics of its interaction with substrate DNA containing an abasic site (AP DNA). The affinity of hAPE binding toward DNA increased as much as 6-fold after replacing a single adenine (equilibrium dissociation constant, K(D), 5.3 nm) with an AP site (K(D), 0.87 nm). The enzyme-substrate complex formation appears to be thermodynamically stabilized and favored by a large change in Gibbs free energy, DeltaG degrees (-50 kJ/mol). The latter is supported by a high negative change in enthalpy, DeltaH degrees (-43 kJ/mol) and also positive change in entropy, DeltaS degrees (24 J/(K mol)), and thus the binding process is spontaneous at all temperatures. Analysis of kinetic parameters reveals small enthalpy of activation for association, DeltaH degrees++(ass) (-17 kJ/mol), and activation energy for association (E(a), -14 kJ/mol) when compared with the enthalpy of activation for dissociation, DeltaH degrees++(diss) (26 kJ/mol), and activation energy in the reverse direction (E(d), 28 kJ/mol). Furthermore, varying concentration of KCl showed an increase in binding affinity at low concentration but complete abrogation of the binding at higher concentration, implying the importance of hydrophobic, but predominantly ionic, forces in the Michaelis-Menten complex formation. Thus, low activation energy and the enthalpy of activation, which are perhaps a result of dipole-dipole interactions, play critical roles in AP site binding of APE.     

Temperature dependence of the dissociation rate constants for the nonactivated (molybdate-stabilized) and activated progesterone receptor-hormone complexes from the chick oviduct

Both the nonactivated and activated forms of the chick oviduct cytosol progesterone receptor-hormone complexes displayed first-order dissociation kinetics at temperatures between 0 and 25 degrees C. The rate constant was always 2-3-times greater for the nonactivated than for the activated complex. The thermodynamic parameters calculated from the Eyring plot for the nonactivated and activated forms, respectively, were: delta H+ = 28.6 +/- 0.2 and 29.9 +/- 1.5 kcal/mol; -T delta S+ = 7.4 +/- 0.6 and 7.7 +/- 1.6 kcal/mol; and delta G+ = 21.3 +/- 0.5 and 22.1 +/- 0.1 kcal/mol. These values suggest that activation results in an increase in enthalpy of the ligand-receptor interaction, thus stabilizing the complex. The dissociation rate constants for the native complex obtained by two different experimental approaches, namely, isotope dilution ('chase') and dissociation against charcoal, indicated the absence of cooperativity in the receptor-ligand binding.     

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)     

Effect of NaCl addition on nanosecond O2 escaping reaction of myoglobin: evidences for the transition of myoglobin dynamic structure at 20 degrees C.

We studied the nanosecond (ns) geminate O2 escape reaction from the protein interior of myoglobin (Mb) to the solvent phase in the temperature range of 5-40 degrees C containing 0-0.1 M NaCl. In the flash photolysis experiments, we found that both the rate constant, kout, and its Arrhenius plot changed upon the variation of the NaCl concentration. In particular, it was noteworthy that the Arrhenius plot of kout dramatically changed in its slope, keeping the break at 20 degrees C, upon the addition of NaCl, indicating that the thermodynamic parameters such as an enthalpy of activation (delta H not equal to) and an entropy of activation (delta S not equal to) are different between above and below 20 degrees C, and that they are further altered upon the NaCl addition to the sample solution. From these results, we suggested that the Mb dynamic structure in the ns geminate O2 escape reaction is sensitively regulated by the interaction of the protein surface and the salt. The present study also showed that an inconsistency of the Arrhenius plot of kout between Chatfield et al. ((1990) J. Am. Chem. Soc. 112, 4680-4687) and us ((1990) J. Biol. Chem. 265, 18823-18828) is probably due to the difference in the solution condition.