Inhibition of lipases by proteins. A kinetic study with dicaprin monolayers

We report further investigations on protein inhibition of pancreatic and microbial lipases carried out with the monolayer technique. When beta-lactoglobulin A, melittin, serum albumin, myoglobin, and a protein inhibiting lipase from soybean were preincubated with a dicaprin film at a surface pressure of 35 dynes/cm, no activity was detected with horse pancreatic or Rhizopus delemar lipases. By contrast, Rhizopus arrhizus and Geotrichum candidum lipase activities were not impaired under the same conditions. Experiments using mixed lipid-protein film transfer clearly show that the inhibition of pancreatic lipase is due to the protein associated with lipid and not caused by direct protein-enzyme interaction in the aqueous phase. Three parameters were used to determine the surface properties of the various proteins at the dicaprin/water interface; namely, the initial rate of surface pressure increase, (delta pi/delta t)t = 0, the maximal surface pressure increase, delta pi max, and the critical surface pressure, pi c. A positive correlation was observed between values of (delta pi/delta t)t = 0 of proteins and their respective capacity to inhibit pancreatic and R. delemar lipases. By contrast, there was no apparent correlation with the two other parameters, delta pi max or pi c.     

Stacking interactions of nucleobases: NMR-investigations. I. Self association of N6, N9-dimethyladenine and N6-dimethyl-N9-ethyladenine.

The selfassociation of N6,N9-dimethyladenine and N6-dimethyl-N9-ethyladenine has been studied by means of NMR technique. The thermodynamic quantities have been calculated using an isodesmic NMR model with three NMR parameters (the monomer shift deltaM and two complex shifts delta2 and delta3). The dependence of the thermodynamic quantities on the NMR parameters is discussed. Special attention is given to the determination of deltaM and its temperature dependence. Calculations with delta3 = 2 - delta2 and deltaM taken independently of temperature result in an average entropy deltaS = - 17.9 +/- 1.8 e.u. for N6,N9-dimethyladenine and deltaS = - 16.7 +/- 1.7 e.u. for N6-dimethyl-N9-ethyladenine and in an average enthalpy deltaH = - 7.2 +/- 0.6 kcal - mol-1 for both substances investigated.     

Thermodynamics and modulation of progesterone microcompartmentation and hydrophobic interaction with cytochrome P450XVII based on quantification of local ligand concentrations in a complex multi-component system

An approach towards the determination of hydrophobic ligand distribution in endoplasmic reticulum membrane suspensions, and of hydrophobic ligand interaction with membrane-anchored proteins, based on calculations of local ligand pools, is presented. Rat testicular microsomes containing cytochrome P450XVII (P450XVII) were used as the model system and considered as consisting of three compartments, i.e. membrane lipid phase, aqueous phase and the ligand-binding protein, P450XVII. Combinations of spectrophotometry, ultracentrifugation and equilibrium dialysis were used to quantify progesterone concentrations in each of the three compartments, as well as partition coefficients, Kp. Since the substrate-access channel of P450XVII is likely to face the membrane-lipid phase, corrected spectral dissociation constants, Ks(corr), were calculated on the basis of free, i.e. not enzyme-bound, progesterone concentrations in the membrane compartment. Modulation of individual components and construction of more complex systems demonstrated the validity of this concept for analysis of multicompartment systems. Although ligand distribution was considerably affected by both ligand and membrane concentrations, Kp and Ks(corr) values were found to be independent of both parameters; Kp values amounted to 1920 and 3120, and Ks(corr) values amounted to 260 microM and 96 microM at 4 degrees C and 25 degrees C, respectively. Thermodynamic parameters delta H, delta S and delta G were calculated from Van't Hoff plots for progesterone partition into the membrane compartment, and for progesterone binding to P450XVII. Both of these processes were entropy dominated, and free energy changes amounted to about -18 kJ/mol for Kp and -20 kJ/mol for Ks(corr). Modification of P450XVII by gonadotropin-induced down-regulation, and by addition of a competitive inhibitor (estradiol) had no effect on progesterone partition. Consideration of Kp = 310 for estradiol allowed the determination of a corrected K1 = 3.09 mM. Modification of the membrane-lipid phase by detergents affected progesterone-P450XVII interaction solely by modulation of Kp; modification of the aqueous phase by addition of bovine serum albumin as a fourth compartment acted solely via additional steroid attraction. This model system therefore stresses the relevance of the local environment of membrane-bound enzymes or receptors for quantification of their interaction with substrates or ligands.     

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).     

Kinetic study of the activation process of frog epidermis pro-tyrosinase by trypsin

1. The rate of tyrosinase formation has been calculated by coupling the activatory process of frog epidermis pro-tyrosinase by trypsin to the oxidation of L-DOPA to dopachrome. Under certain conditions ([trypsin]/[pro-tyrosinase] greater than or equal to 300), the lag period of the coupled reactions, tau, is independent of trypsin concentration. 2. The specific rate constant of tyrosinase formation at different temperatures has been calculated, ranging from 0.025 sec-1, at 5 degrees C to 0.248 sec-1, at 30 degrees C. 3. Thermodynamic parameters of the activatory process (delta G not equal to = + 18.5 kcal/mol; delta H not equal to = + 14.8 kcal/mol; delta S not equal to = -12.4 e.u.; Ea = + 15.3 kcal/mol), have been determined by the study of the system at different temperatures. These values are characteristic for a normal chemical reaction. 4. From these kinetic data, the order of products formation in the proteolytic step, can be determined, active tyrosinase being the last product released.     

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)     

Volumetric properties for the monosaccharide (D-xylose, D-arabinose, D-glucose, D-galactose)-NaCl-water systems at 298.15 K

Densities have been measured for monosaccharide (D-xylose, D-arabinose, D-glucose and D-galactose)-NaCl-water solutions at 298.15 K. These data have been used to determine the apparent molar volumes of these saccharides and NaCl in the studied solutions. Infinite-dilution apparent molar volumes for the saccharides (V0(phi,S)) in aqueous NaCl and those for NaCl (V0(phi,E)) in aqueous saccharide solutions have been evaluated, together with the standard transfer volumes of the saccharides (delta(t) V0S) from water to aqueous NaCl and of NaCl (delta(t) V0E) from water to aqueous saccharide solutions. It is shown that the delta(t) V0S and delta (t) V0E values are positive and increase with increasing co-solute molalities. Volumetric parameters indicating the interactions of NaCl with saccharides in water have been obtained, respectively, by using transfer volumes of the saccharides and NaCl, and the resulting values are in good agreement with each other within experimental error. The interactions between saccharides and NaCl are discussed in terms of the structural interaction model and the stereochemistry of the saccharide molecules in water.     

AMP interaction sites in glycogen phosphorylase b. A thermodynamic analysis

The binding of AMP to activator site N and to inhibitor site I in glycogen phosphorylase b has been characterized by calorimetry, potentiometry and ultracentrifugation in the pH range 6.5-7.5 at 25 degrees C (mu = 0.1). Calorimetric titration data of phosphorylase b with adenosine 5'-phosphoramidate are also reported at pH 6.9 (T = 25 degrees C, mu = 0.1). Calorimetric curves have been analyzed on the basis of potentiometric and sedimentation velocity results to determine thermodynamic quantities for AMP binding to the enzyme. The comparison of calorimetric titration data of AMP and adenosine 5'-phosphoramidate at pH 6.9 supports the hypothesis previously suggested that the dianionic phosphate form of the nucleotide preferentially binds to the allosteric activator site. The thermodynamic parameters for AMP binding to site N are as follows: delta G0 = -22 kJ mol-1, delta H0 = -34 kJ mol-1 and delta S0 = -40 J mol-1 K-1. The binding of the nucleotide to site I was found to be strongly dependent on the pH. This behaviour may be explained in terms of coupled protonations of three groups having pKa values of 6.0, 6.0 and 6.1 in the unbound form and 7.0, 7.5 and 7.2 in the enzyme-nucleotide complex. The thermodynamic parameters for nucleotide binding to site I for the enzymatic form in which all the modified groups are completely deprotonated or protonated have been calculated to be: delta G0 = -7.7 kJ mol-1, delta H0 = -28 kJ mol-1 and delta S0 = -68 J mol-1 K-1 and delta G0 = -28 kJ mol-1, delta H0H = -10 kJ mol-1 and delta S0H = 61 J mol-1 K-1, respectively. These results suggest that attractive dispersion forces are of primary significance for AMP binding to activator site N, although electrostatic interactions act as a stabilizing factor in the nucleotide binding. The protonation states of those residues of which the pKa values are modified by AMP binding to site I highly influence the thermodynamic parameters for the nucleotide binding to this site.     

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.     

Binding of the recombinant proteinase inhibitor eglin c, of the soybean Bowman-Birk proteinase inhibitor and of its chymotrypsin and trypsin inhibiting fragments to Leu-proteinase, the leucine specific serine proteinase from spinach (Spinacia oleracea L.) leaves: 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 (eglin c), of the soybean Bowman-Birk proteinase inhibitor (BBI) and of its chymotrypsin and trypsin inhibiting fragments (F-C and F-T, respectively) to Leu-proteinase, the leucine specific serine proteinase from spinach (Spinacia oleracea L.) leaves, has been investigated. On lowering the pH from 9.5 to 4.5, values of Ka (at 21 degrees C) for complex formation decrease thus reflecting the acidic pK-shift of the hystidyl catalytic residue from approximately 6.9, in the free Leu-proteinase, to approximately 5.1, in the enzyme: inhibitor adducts. At pH 8.0, values of the apparent thermodynamic parameters for the proteinase:inhibitor complex formation are: Leu-proteinase:eglin c-Ka = 2.2 x 10(11) M-1, delta G degree = -64 kJ/mol, delta H degree = +5.9 kJ/mol, and delta S degree = +240 kJ/molK; Leu-proteinase:BBI-Ka = 3.2 x 10(10) M-1, delta G degree = -59 kJ/mol, delta H degree = +8.8 kJ/mol, and delta S degree = +230 J/molK; and Leu-proteinase:F-C-Ka = 1.1 x 10(6) M-1, delta G degree = -34 kJ/mol, delta H degree = +18 J/mol, and delta S degree = +180 J/molK (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 explored, i.e. between 10.0 degrees C and 40.0 degrees C).(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of rabbit secretory component with rabbit IgA dimer.

Secretory component (SC), a glycoprotein with an apparent molecular weight of approximately 80,000, has been isolated from rabbit milk and found to be heterogenous in size and charge. Functionally intact IgA dimer has been dissociated from milk secretory IgA using a chaotropic agent and further purified to homogeneity. The interaction between SC and IgA dimer is a reversible time- and temperature-dependent process. At 23 degrees C, the association rate constant (2.4 x 10(5) M-1 min-1) and the dissociation rate constant (1.8 x 10(-3) min-1) have been measured independently and the affinity constant based on these rates (1.3 x 10(8) M-1) is similar to that calculated from Scatchard plots (1.9 x 10(8) M-1). One class of binding sites has been estimated from Scatchard plots in spite of the observed heterogeneity of SC. The interaction is tighter at low temperatures because the decrease in dissociation rate is greater than the decrease in association rate. The thermodynamic calculations reveal a delta G of -11.0 kcal . mol-1, a delta H of -8.9 kcal . mol-1 and a delta S of +7.0 cal. mol-1 degree-1. The pH range over which interaction occurs is rather large (5 to 8) with no significant differences in apparent Ka.     

Dipolar NMR relaxation of nonprotonated aromatic carbons in proteins. Structural and dynamical effects.

The crystal structure and a 96-ps molecular dynamics simulation used to analyze structural and motional contributions to spin-lattice (T1) relaxation times of phenylalanine and tyrosine C gamma carbons of the pancreatic trypsin inhibitor. The H beta and H delta protons geminal to C gamma are calculated to account for approximately 80% of the dipolar relaxation for each residue. Experimental T1 values for the phenylalanine residues obtained at 25 MHz are observed to be 15-25% longer than estimates based on the rigid crystal structure. It is shown how an increase in T1 can be related to order parameters for the picosecond motional averaging of the important C,H dipolar interactions, and how these order parameters can be calculated from a protein molecular dynamics trajectory.     

Simple kinetic model for the study of lactate metabolic adaptation to exercise in sportsmen routine evaluation

The interindividual specific lactate metabolic adaptation to exercise has been studied. A simple kinetic model was used which did not require labelled molecules. An one open compartment model with a first-order release rate constant described the release of lactate from the muscle. Six volunteers performed five times the same session: pedaling as long as possible at 400 W and 110 rpm. The lactate concentration was measured along the 60 min of recovery. The theoretical curve corresponding to the model was fitted to the experimental data using a non-linear regression method. The values of the following kinetic parameters were obtained: elimination rate constant (ke), release rate constant (ka), apparent amount released into the compartment divided by the volume of distribution (FQo/Vd) and area under the lactate concentration-time curve (AUC). Two way-ANOVA, Scheffé test and discriminant analysis were used to test the statistical significance of these parameters. No significant intra-individual variations were shown. Significant differences were observed between subjects (ke: P = 0.0020; ka: P less than 0.0001; FQo/Vd: P = 0.0002; AUC: P = 0.0395). A correlation was also found between FQo/Vd and ke (r = 0.72; P less than 0.001). In conclusion, the computed parameters provided by the model are sufficient to discriminate and characterize the metabolic response of each subject after short and intensive exercises.     

Study of the enzymatic hydrolysis of a phosphonic ester using microcalorimetry]

A "Batch" microcalorimeter is used at 30 degrees C for the study of the hydrolysis of 4-nitro-phenylphenylphosphonate with a calf-intestinal phosphonate esterase, in a tris buffer, pH 8. The yield of enzymatic hydrolysis is estimated by spectrophotometric determination of the p--nitrophenol evolved; we have then calculated the apparent molar enthalph of the reaction. (delta Happ = -72,2 kj. mol-1). Phenylphosphonic acid, the second reaction product, is not transphosphonylated on tris. The second acidity of phenylphosphonic acid was studied at 30 degrees C by sodium hydroxide electrotitration (pKa2 = 7,13) and by "Flow" microcalorimetry (delta Hionization = 19,8 kj.mol-1). In the same manner at 30 degrees C, we measured the heat of ionization of p-nitrophenol (delta Hionization = 26,75 kj.mol-1). These findings allow a calculation for the actual heat of hydrolysis of 4-nitro-phenyl-phenylphosphonate (delta Hrho = -29,7 kj.mol-1).     

Differential scanning calorimetry study of reversible, partial unfolding transitions in dodecameric glutamine synthetase from Escherichia coli.

Partial unfolding of dodecameric glutamine synthetase (GS) from Escherichia coli has been studied by differential scanning calorimetry (DSC). A single endotherm (tm = 51.6 +/- 0.1 degrees C and delta Hcal = 211 +/- 4 kcal/mol of enzyme) was observed in DSC experiments with Mn.GS in the presence of 1.0 mM free Mn2+ and 100 mM KCl at pH 7. The dodecameric structure of Mn.GS was retained throughout heating cycles, and thermal transitions were reversible as shown by rescans [with 6-18 mg of GS (Mr 622,000) from 15 to 68 degrees C at 20-60 degrees C/h] and by greater than 93% recovery of activity. A cooperative ratio delta Hcal/delta HvH of 1.6 +/- 0.1 and deconvolution analysis show two cooperative units (two-state transitions): t1 = 50.4 and t2 = 51.7 degrees C; the ratio of the relative sizes of thermally labile domains is approximately 1:2 as judged by delta H2/delta H1 approximately equal to 2. However, the thermally induced overall enthalpy change (0.34 cal/g) for GS dodecamer is only 5-10% of that for thermal unfolding of small globular proteins at 50 degrees C. The t1 and t2 values from deconvolutions of DSC data agree with t0.5 values previously calculated from spectral measurements of temperature-induced exposures of approximately 0.7 of 2 Trp and approximately 2 of 17 Tyr per subunit, respectively [Shrake et al. (1989) Biochemistry 28, 6281-6294], over a 14 degrees C temperature range using both stabilizing and destabilizing conditions for Mn.GS.(ABSTRACT TRUNCATED AT 250 WORDS)     

中国南方典型地区油-稻轮作条件下大气SO2干沉降速率动态变化

以1998年11月～1999年10月中国科学院红壤生态试验站农田小气候观测站气象梯度资料(温度、风速、气压),计算近地面湍流特征参数(u^*、θ^*、L),然后采样阻力模式计算SO2干沉降速率(Vd)。研究了该地油-稻轮作条件下大气SO₂干沉降Vd动态变化．结果表明,一年中大气SO2干沉降Vd时平均年均值为0．124～0．897cm·s^-1(mean±SE=0．507±0．167cm·s^-1)．一年中大气SO2干沉降Vd存在如下规律性：白天>晚上．3～8月份SO2Vd平均值(0．611cm·s^-1)大于9～12、1～2月份SO2Vd平均值(0．401cm·s^-1)．水稻生长期间(0．605±0．093cm·s^-1)>油菜(0．491±0．166cm·s^-1)>休闲(0．342±0．174cm·s^-1)．     

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)     

Effect of temperature on the creatine kinase equilibrium.

The effect of temperature on the apparent equilibrium constant of creatine kinase (ATP:creatine N-phosphotransferase (EC 2.7.3.2)) was determined. At equilibrium the apparent K' for the biochemical reaction was defined as [formula: see text] The symbol sigma denotes the sum of all the ionic and metal complex species of the reactant components in M. The K' at pH 7.0, 1.0 mM free Mg2+, and ionic strength of 0.25 M at experimental conditions was 177 +/- 7.0, 217 +/- 11, 255 +/- 10, and 307 +/- 13 (n = 8) at 38, 25, 15, and 5 degrees C, respectively. The standard apparent enthalpy or heat of the reaction at the specified conditions (delta H' degree) was calculated from a van't Hoff plot of log10K' versus 1/T, and found to be -11.93 kJ mol-1 (-2852 cal mol-1) in the direction of ATP formation. The corresponding standard apparent entropy of the reaction (delta S' degree) was +4.70 J K-1 mol-1. The linear function (r2 = 0.99) between log10 K' and 1/K demonstrates that both delta H' degree and delta S' degree are independent of temperature for the creatine kinase reaction, and that delta Cp' degree, the standard apparent heat capacity of products minus reactants in their standard states, is negligible between 5 and 38 degrees C. We further show from our data that the sign and magnitude of the standard apparent Gibbs energy (delta G' degree) of the creatine kinase reaction was comprised mostly of the enthalpy of the reaction, with 11% coming from the entropy T delta S' degree term. The thermodynamic quantities for the following two reference reactions of creatine kinase were also determined. [formula: see text] The delta H degree for Reaction 2 was -16.73 kJ mol-1 (-3998 cal mol-1) and for Reaction 3 was -23.23 kJ mol-1 (-5552 cal mol-1) over the temperature range 5-38 degrees C. The corresponding delta S degree values for the reactions were +110.43 and +83.49 J K-1 mol-1, respectively. Using the delta H' degree of -11.93 kJ mol-1, and one K' value at one temperature, a second K' at a second temperature can be calculated, thus permitting bioenergetic investigations of organs and tissues using the creatine kinase equilibria over the entire physiological temperature range.     

Binding of the bovine basic pancreatic trypsin inhibitor (Kunitz) to the 33,000 Mr and 54,000 Mr species of human urokinase: thermodynamic study

The effect of pH and temperature on the apparent association equilibrium constant (Ka) for the binding of the bovine basic pancreatic trypsin inhibitor (BPTI, Kunitz inhibitor) to the 33,000 Mr and 54,000 Mr species of human urokinase (EC 3.4.21.31) has been investigated. Under all the experimental conditions, values of Ka for BPTI binding to the 33,000 Mr and 54,000 Mr species of human urokinase are identical. On lowering the pH from 9.5 to 4.5, values of Ka (at 21.0 degrees C) for BPTI binding to human urokinase (33,000 Mr and 54,000 Mr species) decrease thus reflecting the acidic pK-shift of the His-57 catalytic residue from 6.9, in the free enzyme, to 5.1, in the proteinase:inhibitor complex. At pH 8.0, values of the apparent thermodynamic parameters for BPTI binding to human urokinase (33,000 Mr and 54,000 Mr species) are: Ka = 4.9 x 10(4) M-1, delta G degree = -6.3 kcal/mol, and delta S degree = -37 entropy units (all at 21.0 degrees C); and delta H degree = +4.6 kcal/mol (temperature independent over the explored range, from 5.0 degrees C to 45.0 degrees C). Thermodynamics of BPTI binding to human urokinase (33,000 Mr and 54,000 Mr species) have been analyzed in parallel with those of related serine (pro)enzyme Kazal- and /Kunitz-type inhibitor systems. Considering the known molecular models, the observed binding behaviour of BPTI to human urokinase (33,000 Mr and 54,000 Mr species) was related to the inferred stereochemistry of the proteinase/inhibitor contact region.     

The vnd/NK-2 homeodomain: thermodynamics of reversible unfolding and DNA binding for wild-type and with residue replacements H52R and H52R/T56W in helix III

The conformational stabilities of the vnd (ventral nervous system defective)/NK-2 homeodomain [HD(wt); residues 1-80 that encompass the 60-residue homeodomain] and those harboring mutations in helix III of the DNA recognition site [HD(H52R) and HD(H52R/T56W)] have been investigated by differential scanning calorimetry (DSC) and ellipticity changes at 222 nm. Thermal unfolding reactions at pH 7.4 are reversible and repeatable in the presence of 50-500 mM NaCl with DeltaC(p) = 0.52 +/- 0.04 kcal K(-1) mol(-1). A substantial stabilization of HD(wt) is produced by 50 mM phosphate or by the addition of 100-500 mM NaCl to 50 mM Hepes, pH 7.4, buffer (from T(m) = 35.5 degrees C to T(m) 43-51 degrees C; DeltaH(vH) congruent with 47 +/- 5 kcal mol(-1)). The order of stability is HD(H52R/T56W) > HD(H52R) > HD(wt), irrespective of the anions present. Progress curves for ellipticity changes at 222 nm as a function of increasing temperature are fitted well by a two-state unfolding model, and the cooperativity of secondary structure changes is greater for mutant homeodomains than for HD(wt) and also is increased by adding 100 mM NaCl to Hepes buffer. A 33% quench of the intrinsic tryptophanyl residue fluorescence of HD(wt) by phosphate binding (K(D)' = 2.6 +/- 0.3 mM phosphate) is reversed approximately 60% by DNA binding. Thermodynamic parameters for vnd/NK-2 homeodomain proteins binding sequence-specific 18 bp DNA have been determined by isothermal titration calorimetry (10-30 degrees C). Values of DeltaC(p) are +0.25, -0.17, and -0.10 +/- 0.04 kcal K(-1) mol(-1) for HD(wt), HD(H52R), and HD(H52R/T56W) binding duplex DNA, respectively. Interactions of homeodomains with DNA are enthalpically controlled at 298 K and pH 7.4 with corresponding DeltaH values of -6.6 +/- 0.5, -10.8 +/- 0.1, and -9.0 +/- 0.6 kcal mol(-1) and DeltaG' values of -11.0 +/- 0.1, -11.0 +/- 0.1, and -11.3 +/- 0.3 kcal mol(-1) with a binding stoichiometry of 1.0 +/- 0.1. Thermodynamic parameters for DNA binding are not predicted from homeodomain structural changes that occur upon complexing to DNA and must reflect also solvent and possibly DNA rearrangements.