Thermodynamics of the binding of galactopyranoside derivatives to the basic lectin from winged bean (Psophocarpus tetrogonolobus).

The thermodynamics of the binding of D-galactopyranoside (Gal), 2-acetamido-2-deoxygalactopyranoside (GalNAc), methyl-alpha-D-galactopyranoside, and methyl-beta-D-galactopyranoside to the basic agglutinin from winged bean (WBAI) in 0.02 M sodium phosphate and 0.15 M sodium chloride buffer have been investigated from 298.15 to 333.15 K by titration calorimetry and at the denaturation temperature by differential scanning calorimetry (DSC). WBAI is a dimer with two binding sites. The titration calorimetry yielded single-site binding constants ranging from 0.56 +/- 0.14 x 10(3) M-1 for Gal at 323.15 K to 7.2 +/- 0.5 x 10(3) M-1 for GalNAc at 298.15 K and binding enthalpies ranging from -28.0 +/- 2.0 kJ mol-1 for GalNAc at 298.15 K to -14.3 +/- 0.1 kJ mol-1 for methyl-beta-D-galactopyranoside at 322.65 K. The denaturation transition consisted of two overlapping peaks over the pH range 5.6-7.4. Fits of the differential scanning calorimetry data to a two-state transition model showed that the low temperature transition (341.6 +/- 0.4 K at pH 7.4) consisted of two domains unfolding as a single entity while the higher temperature transition (347.8 +/- 0.6 K at pH 7.4) is of the remaining WBAI dimer unfolding into two monomers. Both transitions shift to higher temperatures and higher calorimetric enthalpies with increase in added ligand concentration at pH 7.4. Analysis of the temperature increase as a function of added ligand concentration suggests that one ligand binds to the two domains unfolding at 341.6 +/- 0.6 K and one ligand binds to the domain unfolding at 347.8 +/- 0.6 K.     

Energetic differences between the specific binding of a 40 bp DNA duplex and the lac promoter to lac repressor protein

The energetics of LRP binding to a 104 bp lac promoter determined from ITC measurements were compared to the energetics of binding to a shorter 40 bp DNA duplex with the 21 bp promoter binding site sequence. The promoter binding affinity of 2.47 +/- 0.0 1x 10(7) M(-1) was higher than the DNA binding affinity of 1.81 +/- 0.67 x 10(7) M(-1) while the binding enthalpy of -804 +/- 41 kJ mol(-1) was lower than that of the DNA binding enthalpy of -145 +/- 16 kJ mol(-1) at 298.15 K. Both the promoter and DNA binding reactions were exothermic in phosphate buffer but endothermic in Tris buffer that showed the transfer of four protons to LRP in the former reaction but only two in the latter. A more complicated dependence of these parameters on temperature was observed for promoter binding. These energetic differences are attributable to additional LRP-promoter interactions from wrapping of the promoter around the LRP.     

Interaction of cytidine 3'-monophosphate and uridine 3'-monophosphate with ribonuclease a at the denaturation temperature

Differential scanning calorimetry (DSC) measurements were performed on the thermal denaturation of ribonuclease a and ribonuclease a complexed with an inhibitor, cytidine or uridine 3'-monophosphate, in sodium acetate buffered solutions. Thermal denaturation of the complex results in dissociation of the complex into denatured ribonuclease a and free inhibitor. Binding constants of the inhibitor to ribonuclease a were determined from the increase in the denaturation temperature of ribonuclease a in the complexed form and from the denaturation enthalpy of the complex. Binding enthalpies of the inhibitor to ribonuclease a were determined from the increase in the denaturation enthalpy of ribonuclease a complexed with the inhibitor. For the cytidine inhibitor in 0.2 M sodium acetate buffered solutions, the binding constants increase from 87 +/- 8 M-1 (pH 7.0) to 1410 +/- 54 M-1 (pH 5.0), while the binding enthalpies increase from 17 +/- 13 kJ mol-1 (pH 4.7) to 79 +/- 15 kJ mol-1 (pH 5.5). For the uridine inhibitor in 0.2 M sodium acetate buffered solutions, the binding constants increase from 104 +/- 1 M-1 (pH 7.0) to 402 +/- 7 M-1 (pH 5.5), while the binding enthalpies increase from 16 +/- 5 kJ mol-1 (pH 6.0) to 37 +/- 4 kJ mol-1 (pH 7.0). The binding constants and enthalpies of the cytidine inhibitor in 0.05 M sodium acetate buffered solutions increase respectively from 328 +/- 37 M-1 (pH 6.5) to 2200 +/- 364 M-1 (pH 5.5) and from 22 kJ mol-1 (pH 5.5) to 45 +/- 7 kJ mol-1 (pH 6.5). the denaturation transition cooperativities of the uncomplexed and complexed ribonuclease a were close to unity, indicating that the transition is two state with a stoichiometry of 1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermodynamics of the hydrolysis of adenosine 5'-triphosphate to adenosine 5'-diphosphate

The enthalpy of hydrolysis of the enzyme-catalyzed (heavy meromyosin) conversion of adenosine 5'-triphosphate (ATP) to adenosine 5'-diphosphate (ADP) and inorganic phosphate has been investigated using heat-conduction microcalorimetry. Enthalpies of reaction were measured as a function of ionic strength (0.05-0.66 mol kg-1), pH (6.4-8.8), and temperature (25-37 degrees C) in Tris/HCl buffer. The measured enthalpies were adjusted for the effects of proton ionization and metal ion binding, protonation and interaction with the Tris buffer, and ionic strength effects to obtain a value of delta H0 = -20.5 +/- 0.4 kJ mol-1 at 25 degrees C for the process, ATP4-(aq) + H2O(l) = ADP3-(aq) + HPO2-4(aq) + H+(aq) where aq is aqueous and l is liquid. Heat measurements carried out at different temperatures lead to a value of delta C0p = -237 +/- 30 J mol-1 K-1 for the above process.     

Calorimetric studies of the ADP binding to myosin subfragment 1, heavy meromyosin, and to myosin filaments.

A calorimetric titration method was used to study the ADP binding to the chymotryptic subfragments of myosin, heavy meromyosin (HMM) and myosin subfragment 1 (S-1), and to myosin aggregated into filaments at low ionic strength. The binding constant (K) and heat of reaction (deltaH, kiloJoules (moles of ADP bound)-1) were determined. For HMM in 0.5 M KCl, 0.01 M MgCl2, 0.02 M Tris (pH 7.8) at 12 degrees, log K = 5.92 +/- 0.13 and deltaH = -70.9 +/- 3.6 kJ mol-1. These results agree with our previous findings for myosin in 0.5 M KCl at 12 degrees. When the KCl concentration was reduced to 0.1 M, the binding constant did not change significantly (log K = 6.09 +/- 0.06) but the binding was more exothermic (deltaH = -90.1 +/- 3.3 kJ mol-1). Similar results were obtained for myosin filaments in 0.1 M KCl and also for both the isoenzymes of S-1(S-1(A1) and S-1(A2) in 0.1 M KCl. In 0.5 M KCl, the binding curves suggest that about one ADP is bound per active site, but as 0.1 M KCl, the apparent stoichiometry drops from 0.7 to 0.75. The most probable explanation is that there is some site heterogeneity which is more evident at lower ionic strength.     

A thermodynamic study of the interaction of tubulin with colchicine site ligands

The bicyclic colchicine analogue 2-methoxy-5-(2',3',4'-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-on e (MTC) has been used to study the thermodynamics of specific ligand binding to the colchicine site of tubulin, employing isothermal reaction microcalorimetry. The binding of MTC to purified calf brain tubulin, in 10 mM sodium phosphate buffer, pH 7.0, is characterized by delta H degree = -19 +/- 1 kJ.mol-1, delta G degree = -31.8 +/- 0.6 kJ.mol-1, and delta S degree = 43 +/- 5 J.mol-1.K-1 at 298 K, with a slight variation in the temperature range from 283 to 308 K. The binding thermodynamics of colchicine and allocolchicine are similar to MTC under the conditions examined, suggesting related molecular interactions of the three ligands with the protein binding site. The standard enthalpy changes of binding of colchicine and MTC at 308 K coincide within experimental error. Therefore the more favorable free energy change of binding of colchicine must come from a larger binding entropy change (by about 20 J.mol-1.K-1). This difference could be attributed to the presence of the middle ring of colchicine, which is absent in MTC. Consistently, a similar entropy change is observed by the comparison of allocolchicine to MTC binding at several temperatures. In addition, allocolchicine binding is about 6 kJ.mol-1 less exothermic than MTC binding, which could be attributed to the presence in allocolchicine of a substituted phenyl ring instead of the colchicine-MTC tropolone ring. The present results and analysis are fully compatible with the previously proposed bifunctional binding of colchicine and MTC (through their trimethoxybenzene and tropolone moieties) to a bifocal protein binding site, and also with a partial immobilization of intramolecular rotation of MTC upon binding, which in colchicine is already constrained by its middle ring (Andreu, J. M., Gorbunoff, M. J., Lee, J. C., and Timasheff, S. (1984) Biochemistry 23, 1742-1752).     

An investigation of the equilibria between aqueous ribose, ribulose, and arabinose

The thermodynamics of the equilibria between aqueous ribose, ribulose, and arabinose were investigated using high-pressure liquid chromatography and microcalorimetry. The reactions were carried out in aqueous phosphate buffer over the pH range 6.8-7.4 and over the temperature range 313.15-343.75 K using solubilized glucose isomerase with either Mg(NO3)2 or MgSO4 as cofactors. The equilibrium constants (K) and the standard state Gibbs energy (delta G degrees) and enthalpy (delta H degrees) changes at 298.15 K for the three equilibria investigated were found to be: ribose(aq) = ribulose(aq) K = 0.317, delta G degrees = 2.85 +/- 0.14 kJ mol-1, delta H degrees = 11.0 +/- 1.5 kJ mol-1; ribose(aq) = arabinose(aq) K = 4.00, delta G degrees = -3.44 +/- 0.30 kJ mol-1, delta H degrees = -9.8 +/- 3.0 kJ mol-1; ribulose(aq) = arabinose(aq) K = 12.6, delta G degrees = -6.29 +/- 0.34 kJ mol-1, delta H degrees = -20.75 +/- 3.4 kJ mol-1. Information on rates of the above reactions was also obtained. The temperature dependencies of the equilibrium constants are conveniently expressed as R in K = -delta G degrees 298.15/298.15 + delta H degrees 298.15[(1/298.15)-(1/T)] where R is the gas constant (8.31441 J mol-1 K-1) and T the thermodynamic temperature.     

Comparison of the binding of Na+ and Ca2+ to bovine alpha-lactalbumin

alpha-Lactalbumin is a metal-binding protein which binds Ca2+- and Na+-ions competitively to one specific site, giving rise to a large conformational change of the protein. For this reason, the enthalpy change of binding Ca2+ to apo-alpha-lactalbumin (delta Ho) is strongly dependent on the concentration of Na+ ions in the medium. From that relationship a molar enthalpy of -145 +/- 3 kJ X mol-1 is calculated for the Ca2+-binding at pH 7.4 and 25 degrees C, while a delta Ho of -5 +/- 3 kJ X mol-1 is found to substitute a complexed Na+ by a Ca2+-ion. These measurements also allowed us to calculate a binding constant for Na+ of 195 +/- 18 M-1. The molar enthalpy of Na+-loading was found to be -142 +/- 3 kJ X mol-1, a value very close to delta Ho of the binding of Ca2+ to alpha-lactalbumin. Both enthalpy changes in binding Ca2+ and Na+ are independent of the protein concentration. These exothermic values are in agreement with the hypothesis that both Na+- and Ca2+-ions are able to induce the same conformational change in alpha-lactalbumin upon which hydrophobic regions are removed from the solvent, yielding a less hydrophobic protein. The latter is confirmed by means of affinity measurements of the hydrophobic fluorescent probe 4,4'-bis[1-(phenylamino)-8-naphthalene sulphonate](bis-ANS) to alpha-lactalbumin. The association constant (Ka) decreased from (6.6 +/- 0.5) X 10(4) M-1 in the absence of NaCl to (2.7 +/- 0.2) X 10(4) M-1 in 75 mM NaCl, while the maximum intensity (Imax) of the binary bis-ANS-alpha-lactalbumin complex remained constant at 0.44 +/- 0.02 (arbitrary units). The Ka value of bis-ANS for Ca2+-alpha-lactalbumin was determined at (1.7 +/- 0.2) X 10(4) M-1 and Imax was 0.43 +/- 0.02 (arbitrary units). The difference in hydrophobicity between the two conformational states of the protein was further demonstrated by adsorption experiments of both conformers to phenyl-Sepharose. Apo-alpha-lactalbumin, hydrophobically bound to phenyl-Sepharose, can be eluted by adding Ca2- or Na+-solutions.     

Thermodynamics of hydrolysis of disaccharides. Lactulose, alpha-D-melibiose, palatinose, D-trehalose, D-turanose and 3-o-beta-D-galactopyranosyl-D-arabinose

High-pressure liquid chromatography and microcalorimetry have been used to study the thermodynamics of the hydrolysis reactions of a series of disaccharides. The enzymes used to bring about the hydrolyses were: beta-galactosidase for lactulose and 3-o-beta-D-galactopyranosyl-D-arabinose; beta-glucosidase for alpha-D-melibiose; beta-amylase for D-trehalose; isomaltase for palatinose; and alpha-glucosidase for D-turanose. The buffer used was sodium acetate (0.02-0.10 M and pH 4.44-5.65). For the following processes at 298.15 K: lactulose(aq) + H2O(liq) = D-galactose(aq) + D-fructose(aq), K0 = 128 +/- 10 and delta H0 = 2.21 +/- 0.10 kJ mol-1; alpha-D-melibiose(aq) + H2O(liq) = D-galactose(aq) + D-glucose(aq), K0 = 123 +/- 42 and delta H0 = -0.88 +/- 0.50 kJ mol-1; palatinose(aq) + H2O(liq) = D-glucose(aq) + D-fructose(aq), delta H0 = -4.44 +/- 1.1 kJ mol-1; D-trehalose(aq) + H2O(liq) = 2 D-glucose(aq), K0 = 119 +/- 10 and delta H0 = 4.73 +/- 0.41 kJ mol-1; D-turanose(aq) + H2O(liq) = D-glucose(aq) + D-fructose(aq), delta H0 = -2.68 +/- 0.75 kJ mol-1; and 3-o-beta-D-galactopyranosyl-D-arabinose(aq) + H2O(liq) = D-galactose(aq) + D- arabinose(aq),0H0 = 107 +/- 10 and delta H0 = 2.97 +/- 0.10 kJ mol-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermodynamics of isomerization reactions involving sugar phosphates

Thermodynamics of isomerization reactions involving sugar phosphates have been studied using heat-conduction microcalorimetry. For the process glucose 6-phosphate2-(aqueous) = fructose 6-phosphate2- (aqueous), K = 0.285 +/- 0.004, delta Go = 3.11 +/- 0.04 kJ.mol-1, delta Ho = 11.7 +/- 0.2 kJ.mol-1, and delta Cop = 44 +/- 11 J.mol-1.K-1 at 298.15 K. For the process mannose 6-phosphate2- (aqueous) = fructose 6-phosphate2- (aqueous), K = 0.99 +/- 0.05, delta Go = 0.025 +/- 0.13 kJ.mol-1, delta Ho = 8.46 +/- 0.2 kJ.mol-1, and delta Cop = 38 +/- 25 J.mol-1.K-1 at 298.15 K. The standard state is the hypothetical ideal solution of unit molality. An approximate result (-14 +/- 5 kJ.mol-1) was obtained for the enthalpy of isomerization of ribulose 5-phosphate (aqueous) to ribose 5-phosphate (aqueous). The data from the literature on isomerization reactions involving sugar phosphates have been summarized, adjusted to a common reference state, and examined for trends and relationships to each other and to other thermodynamic measurements. Estimates are made for thermochemical parameters to predict the state of equilibrium of the several isomerizations considered herein.     

Thermodynamics of hydrolysis of disaccharides. Cellobiose, gentiobiose, isomaltose, and maltose

The thermodynamics of the enzymatic hydrolysis of cellobiose, gentiobiose, isomaltose, and maltose have been studied using both high pressure liquid chromatography and microcalorimetry. The hydrolysis reactions were carried out in aqueous sodium acetate buffer at a pH of 5.65 and over the temperature range of 286 to 316 K using the enzymes beta-glucosidase, isomaltase, and maltase. The thermodynamic parameters obtained for the hydrolysis reactions, disaccharide(aq) + H2O(liq) = 2 glucose(aq), at 298.15 K are: K greater than or equal to 155, delta G0 less than or equal to -12.5 kJ mol-1, and delta H0 = -2.43 +/- 0.31 kJ mol-1 for cellobiose; K = 17.9 +/- 0.7, delta G0 = -7.15 +/- 0.10 kJ mol-1 and delta H0 = 2.26 +/- 0.48 kJ mol-1 for gentiobiose; K = 17.25 +/- 0.7, delta G0 = -7.06 +/- 0.10 kJ mol-1, and delta H0 = 5.86 +/- 0.54 kJ mol-1 for isomaltose; and K greater than or equal to 513, delta G0 less than or equal to -15.5 kJ mol-1, and delta H0 = -4.02 +/- 0.15 kJ mol-1 for maltose. The standard state is the hypothetical ideal solution of unit molality. Due to enzymatic inhibition by glucose, it was not possible to obtain reliable values for the equilibrium constants for the hydrolysis of either cellobiose or maltose. The entropy changes for the hydrolysis reactions are in the range 32 to 43 J mol-1 K-1; the heat capacity changes are approximately equal to zero J mol-1 K-1. Additional pathways for calculating thermodynamic parameters for these hydrolysis reactions are discussed.     

Thermodynamics of the reactions catalyzed by the multifunctional enzyme complex tryptophan synthase

The intrinsic enthalpy changes (corrected for hydration of D-glyceraldehyde 3-phosphate) for the reactions catalyzed by the alpha and beta 2 subunits of tryptophan synthase from Escherichia coli have been determined calorimetrically. Cleavage of indoleglycerol phosphate (alpha reaction) was found to be associated with a delta H value of 54.0 +/- 2.5 kJ mol-1, while condensation of indole with L-serine (beta reaction) involved -80.3 +/- 4.6 kJ mol-1'. By direct determination of the enthalpy concomitant with the overall synthesis of tryptophan from indoleglycerol phosphate and L-serine an enthalpy value of -13.4 +/- 5.6 kJ mol-1 was observed. In view of the uncertainties of the literature data used for calculation of the hydration contribution, the agreement between the directly measured delta H value of the overall reaction and the sum of the enthalpies of the alpha and beta reactions is fair. Deamination of L-serine, a side reaction catalyzed preferentially by the isolated beta 2 pyridoxal 5'-phosphate2 subunit, was shown to be associated with an enthalpy change of -7.3 +/- 0.4 kJ mol-1.     

Differential scanning calorimetric studies on binding of N-acetyl-D-glucosamine to lysozyme

Differential scanning calorimetric (DSC) measurements were performed on the thermal denaturation of lysozyme and lysozyme complexed with N-acetyl-D-glucosamine (GlcNAc) at pH 5.00 (acetate buffer), 4.25 and 2.25 (Gly-HCl buffer). DSC data have been analyzed to obtain denaturation temperature T(d), enthalpy of denaturation DeltaH(D), heat capacity of denaturation DeltaC(pd) and cooperativity index eta. From these thermodynamic parameters, the binding constant K(L) and enthalpy of binding DeltaH(L), for the weak binding of lysozyme with GlcNAc have been determined. The values of K(L) and DeltaH(L) at pH 5.00 and 298 K are 42 +/- 4 M(-1) and -24 +/- 4 kJ mol(-1), respectively, and agree very well with the experimentally determined values from equilibrium and other studies. The binding constant has also been estimated by simulating the DSC curve with varying values of K(L) (T(d)) until it matches the experimental curve.     

Studies of the cellulolytic system of Trichoderma reesei QM 9414. Binding of small ligands to the 1,4-beta-glucan cellobiohydrolase II and influence of glucose on their affinity

Binding onto cellobiohydrolase II from Trichoderma reesei of glucose, cellobiose, cellotriose, derivatized and analogous compounds, is monitored by protein-difference-absorption spectroscopy and by titration of ligand fluorescence, either at equilibrium or by the stopped-flow technique. The data complete earlier results [van Tilbeurgh, H., Pettersson, L. G., Bhikhabhai, R., De Boeck, H. and Claeyssens, M. (1985) Eur. J. Biochem. 148, 329-334] indicating an extended active center, with putative subsites ABCD. Subsite A specifically complexes with beta-D-glucosides and D-glucose; at 25 degrees C the latter influences the concomitant binding of other ligands at neighbouring sites. For several ligands this cooperative effect for binding (at 0.33 M glucose and temperature range 4-37 degrees C) was characterized by a substantial increase of the enthalpic term (delta delta H = -35 kJ mol-1). Glucose (0.33 M) decreases the association and dissociation rate parameters of 4-methylumbelliferyl beta-D-cellobioside by one order of magnitude: k+ = (3.6 +/- 0.5) x 10(-5) M-1 s-1 versus (5.1 +/- 0.1) x 10(-6) M-1 s-1 (in the absence of glucose) and k- = (1.3 +/- 0.1) s-1 versus (14.0 +/- 0.3) s-1. As deduced from substrate-specificity studies and inhibition experiments, subsite B interacts with terminal non-reducing glucopyranosyl residues of oligomeric ligands and substrates, whereas catalytic (hydrolytic) cleavage occurs between C and D. Association constants 10-100 times higher than those for cellobiose or its glycosides were observed for D-glucopyranosyl-(1----4)-beta-D-xylopyranose and cellobionolactone derivatives, suggesting 'transition-state'-type binding for these ligands at subsite C. Although subsite D can accomodate a bulky chromophoric group (MeUmb) its preference for a glucosyl residue is reflected in the lower binding enthalpy of cellotriose (-34 kJ mol-1) as compared to cellobiose (-28.3 kJ mol-1) and MeUmb(Glc)2 (-11.6 kJ mol-1). This model indicates that oligomeric ligands (substrates) interact through cooperativity of their subunits at the extended binding site of cellobiohydrolase II.     

Calorimetric studies of the interaction of myosin with ADP.

A calorimetric titration method was used to study ADP binding to native myosin. Data were analyzed by assuming that the myosin molecule has n independent and identical sites for ADP binding. The enthalpy change (deltaH), the binding constant (K), and n were determined. In 0.5 M KCl, 0.01 M MgCl2, and 0.02 M Tris/HCl (pH 7.8), we found: at 0 degrees, deltaH = -57.1 +/- 3.2 kJ-mol-1, log K = 6.42 +/- 0.13, n = 1.49 +/- 0.07; at 12 degrees, deltaH = 73.1 +/- 3.2 kJ-mole-1, log K = 6.08 +/- 0.13, and n = 1.74 +/- 0.07. The average heat capacity change on ADP binding to myosin between 0 and 12 degrees is thus -1.4 +/- 0.4 kJ-mol-1-K-1. Reasonably consistent results were obtained at 25 degrees, suggesting ADP binding to myosin is as strongly exothermic as at lower temperatures, although further interpretation of this result seems unwarranted, mainly because of the instability of myosic at this temperature. The number of protons released on binding of ADP to myosin was determined in separate experiments. The value was 0.19 +/- 0.02 at both 0 and 12 degrees. The reaction of protons with Tris thus contributes about -9.5 kJ-mol-1 to the observed heat on ADP binding.     

Binding of anandamide to bovine serum albumin

The endocannabinoid anandamide is of lipid nature and may thus bind to albumin in the vascular system, as do fatty acids. The knowledge of the free water-phase concentration of anandamide is essential for the investigations of its transfer from the binding protein to cellular membranes, because a water-phase shuttle of monomers mediates such transfers. We have used our method based upon the use of albumin-filled red cell ghosts as a dispersed biological "reference binder" to measure the water-phase concentrations of anandamide. These concentrations were measured in buffer (pH 7.3) in equilibrium with anandamide bound to BSA inside resealed human red cell membranes at low molar ratios below one. Data were obtained at 0 degrees C, 10 degrees C, 23 degrees C, and 37 degrees C. The equilibrium dissociation constant (Kd) increases with temperature from 6.87 +/- 0.53 nM at 0 degrees C to 54.92 +/- 1.91 nM at 37 degrees C. Regression analyses of the data suggest that BSA has one high-affinity binding site for anandamide at all four temperatures. The free energy of anandamide binding (DeltaG0) is calculated to -43.05 kJ mol-1 with a large enthalpy (DeltaH0) contribution of -42.09 kJ mol-1. Anandamide has vasodilator activity, and the binding to albumin may mediate its transport in aqueous compartments.     

Thermal stability of PNA/DNA and DNA/DNA duplexes by differential scanning calorimetry

Thermodynamics of the thermal dissociation transitions of 10 bp PNA/DNA duplexes and their corresponding DNA/DNA duplexes in 10 mM sodium phosphate buffer (pH 7.0) were determined from differential scanning calorimetry (DSC) measurements. The PNA/DNA transition temperatures ranged from 329 to 343 K and the calorimetric transition enthalpies ranged from 209 +/- 6 to 283 +/- 37 kJ mol(-1). The corresponding DNA/DNA transition temperatures were 7-20 K lower and the transition enthalpies ranged from 72 +/- 29 to 236 +/- 24 kJ mol(-1). Agreement between the DSC and UV monitored melting (UVM) determined transition enthalpies validated analyzing the UVM transitions in terms of a two-state transition model. The transitions exhibited reversibility and were analyzed in terms of an AB = A + B two-state transition model which yielded van't Hoff enthalpies in agreement with the transition enthalpies. Extrapolation of the transition enthalpies and free energy changes to ambient temperatures yielded more negative values than those determined directly from isothermal titration calorimetry measurements on formation of the duplexes. This discrepancy was attributed to thermodynamic differences in the single-strand structures at ambient and at the transition temperatures, as indicated by UVM measurements on single DNA and PNA strands.     

Thermodynamics of the binding of Streptomyces subtilisin inhibitor to alpha-chymotrypsin

The binding of Streptomyces subtilisin inhibitor (SSI) to alpha-chymotrypsin (CT) (EC 3.4.21.1) was studied by isothermal and differential scanning calorimetry at pH 7.0. Thermodynamic quantities for the binding of SSI to the enzyme were derived as functions of temperature from binding constants (S. Matsumori, B. Tonomura, and K. Hiromi, private communication) and isothermal calorimetric experiments at 5-30 degrees C. At 25 degrees C, the values are delta G degrees b = -29.9 kJ mol-1, delta Hb = +18.7 (+/- 1.3) kJ mol-1, delta S degrees b = +0.16 kJ K-1 mol-1, and delta C p,b = -1.08 (+/- 0.11) kJ mol-1. The binding of SSI to CT is weak compared with its binding to subtilisin [Uehara, Y., Tonomura, B., & Hiromi, K. (1978) J. Biochem. (Tokyo) 84, 1195-1202; Takahashi, K., & Fukada, H. (1985) Biochemistry 24, 297-300]. This difference is due primarily to a less favorable enthalpy change in the formation of the complex with CT. The hydrophobic effect is presumably the major source of the entropy and heat capacity changes which accompany the binding process. The unfolding temperature of the complex is about 7 degrees C higher than that of the free enzyme. The enthalpy and the heat capacity changes for the unfolding of CT were found to be 814 kJ mol-1 and 17.3 kJ K-1 mol-1 at 49 degrees C. The same quantities for the unfolding of the SSI-CT complex are 1183 kJ mol-1 and 39.2 kJ K-1 mol-1 at 57 degrees C.     

Thermodynamic study of yeast phosphoglycerate kinase

Enthalpies of binding of MgADP, MgATP, and 3-phosphoglycerate to yeast phosphoglycerate kinase have been determined by flow calorimetry at 9.95-32.00 degrees C. Combination of these data with published dissociation constants [Scopes, R.K. (1978) Eur. J. Biochem. 91, 119-129] yielded the following thermodynamic parameters for the binding of 3-phosphoglycerate at 25 degrees C: delta Go = -6.76 +/- 0.11 kcal mol-1, delta H = 3.74 +/- 0.08 kcal mol-1, delta So = 35.2 +/- 0.6 cal K-1 mol-1, and delta Cp = 0.12 +/- 0.32 kcal K-1 mol-1. The thermal unfolding of phosphoglycerate kinase in the absence and presence of the ligands listed above was studied by differential scanning calorimetry. The temperature of half-completion, t 1/2, of the denaturation and the denaturational enthalpy are increased by the binding of the ligands, the increase in t 1/2 being a manifestation of Le Chatelier's principle and that in enthalpy reflecting the enthalpy of dissociation of the ligand. Only one denaturational peak was observed under all conditions, and in contrast with the case of yeast hexokinase [Takahashi, K., Casey, J.L., & Sturtevant, J.M. (1981) Biochemistry 20, 4693-4697], no definitive evidence for the unfolding of more than one domain was obtained.