Thermodynamic and kinetic analysis of carbohydrate binding to the basic lectin from winged bean (Psophocarpus tetragonolobus)

A basic lectin (pI approximately 10.0) was purified to homogeneity from the seeds of winged bean (Psophocarpus tetragonolobus) by affinity chromatography on Sepharose 6-aminocaproyl-D-galactosamine. The lectin agglutinated trypsinized rabbit erythrocytes and had a relative molecular mass of 58,000 consisting of two subunits of Mr 29,000. The lectin binds to N-dansylgalactosamine, leading to a 15-fold increase in dansyl fluorescence with a concomitant 25-nm blue shift in the emission maximum. The lectin has two binding sites/dimer for this sugar and an association constant of 4.17 X 10(5) M-1 at 25 degrees C. The strong binding to N-dansylgalactosamine is due to a relatively positive entropic contribution as revealed by the thermodynamic parameters: delta H = -33.62 kJ mol-1 and delta S0 = -5.24 J mol-1 K-1. Binding of this sugar to the lectin shows that it can accommodate a large hydrophobic substituent on the C-2 carbon of D-galactose. Studies with other sugars indicate that a hydrophobic substituent in alpha-conformation at the anomeric position increases the affinity of binding. The C-4 and C-6 hydroxyl groups are critical for sugar binding to this lectin. Lectin difference absorption spectra in the presence of N-acetylgalactosamine indicate perturbation of tryptophan residues on sugar binding. The results of stopped flow kinetics with N-dansylgalactosamine and the lectin are consistent with a simple one-step mechanism for which k+1 = 1.33 X 10(4) M-1 s-1 and k-1 = 3.2 X 10(-2) s-1 at 25 degrees C. This k-1 is slower than any reported for a lectin-monosaccharide complex so far. The activation parameters indicate an enthalpically controlled association process.     

Binding of simple carbohydrates and some of their chromophoric derivatives to soybean agglutinin as followed by titrimetric procedures and stopped flow kinetics

The number of carbohydrate-binding sites of the GalNAc-specific lectin is four per tetramer. The binding parameters of N-acetyl-D-galactosamine and methyl-N-acetyl-alpha-D- galactosaminide , were determined by titrating the perturbation in the absorption spectrum of the protein. For D-galactosides, it was necessary to use p-nitrophenyl-N-acetyl-beta-D- galactosaminide as an indicator in substitution titrations. The association constants K were determined at several temperatures yielding 2.4 X 10(4) M-1 at 25 degrees C with delta H degree' = -45 kJ mol-1 and delta S degree' = -67 J X K-1 mol-1 for methyl-N-acetyl-alpha-D- galactosaminide and 1.0 X 10(3) M-1 at 25 degrees C, delta H degree' = -38 kJ mol-1 and delta S degree' = -69 J X K-1 mol-1 for methyl-alpha-D-galactoside. The increase in K by a factor of 25 caused by the acetamido group is largely enthalpic . Whenever different methods were used to determine the association constant of a given compound, the agreement was excellent. The observed changes in absorption or fluorescence of all chromophoric carbohydrate derivatives used are specific for the binding of carbohydrates. For large aromatic beta- aglycons such as p-nitrophenyl or 4-methylumbelliferyl groups, the increase in K of the N-acetyl-D- galactosaminide moiety is by a factor of 2 or less, but for a large N-5-dimethylaminonaphthalene-1-sulfonyl (dansyl) group this factor is about 20 as compared with the acetyl group. The concomitant 10-fold increase in dansyl fluorescence, also observed with four other GalNAc-binding lectins together with a favorable and large delta S degree' = +60 J X K-1 mol-1 strongly point at the presence of a hydrophobic region in the vicinity of the carbohydrate-binding site. The results of stopped flow kinetics with 4-methylumbelliferyl-N-acetyl-beta-D- galactosaminide and the lectin are consistent with a simple mechanism for which k+ = 1.1 X 10(4) M-1 S-1 and k- = 0.4 S-1 at 25 degrees C. This k- is slower than for any monosaccharide-lectin complex reported so far.     

Analysis of saccharide binding to Artocarpus integrifolia lectin reveals specific recognition of T-antigen (beta-D-Gal(1----3)D-GalNAc)

The binding of Artocarpus integrifolia lectin to N-dansylgalactosamine (where dansyl is 5-dimethylaminonaphthalene-1-sulfonyl) leads to a 100% increase in dansyl fluorescence with a concomitant blue shift in the emission maximum by 10 nm. This binding is carbohydrate-specific and has an association constant of 1.74 X 10(4) M-1 at 20 degrees C. The lectin has two binding sites for N-dansylgalactosamine. The values of -delta H and -delta S for the binding of N-dansylgalactosamine are in the range of values reported for several lectin-monosaccharide interactions, indicating an absence of nonpolar interaction of the dansyl moiety of the sugar with the combining region of the protein. Dissociation of the bound N-dansylgalactosamine from its complex with the lectin and the consequent change in its fluorescence on addition of nonfluorescent sugars allowed evaluation of the association constant for competing ligands. The thermodynamic parameters for the binding of monosaccharides suggest that the OH groups at C-2, C-3, C-4, and C-6 in the D-galactose configuration are important loci for interaction with the lectin. The acetamido group at C-2 of 2-acetamido-2-deoxygalactopyranose and a methoxyl group at C-1 of methyl-alpha-D-galactopyranoside are presumably also involved in binding through nonpolar and van der Waals' interactions. The T-antigenic disaccharide Gal beta 1----3GalNAc binds very strongly to the lectin when compared with methyl-beta-D-galactopyranoside, the beta(1----3)-linked disaccharides such as Gal beta 1----3GlcNAc, and the beta(1----4)-linked disaccharides, N-acetyllactosamine and lactose. The major stabilizing force for the avid binding of T-antigenic disaccharide appears to be a favorable enthalpic contribution. The combining site of the lectin is, therefore, extended. These data taken together suggest that the Artocarpus lectin is specific toward the Thomsen-Friedenreich (T) antigen. There are subtle differences in the overall topography of its combining site when compared with that of peanut (Arachis hypogaea) agglutinin. The results of stopped flow spectrometry for the binding of N-dansylgalactosamine tot he Artocarpus lectin are consistent with a simple single-step bimolecular association and unimolecular dissociation rate processes. The value of K+1 and K-1 at 21 degrees C are 8.1 X 10(5) M-1 s-1 and 50 s-1, respectively. The activation parameters indicate an enthalpy-controlled association process.     

Binding of N-dansylgalactosamine to winged-bean tuber lectin: studies by fluorescence quenching titrations

The winged-bean tuber lectin binds to N-dansyl(5-dimethylaminonaphthalene-1-sulphonic acid)galactosamine, leading to a 12.5-fold increase in dansyl fluorescence with a concomitant 25 nm blue-shift in the emission maximum. The enhancement of fluorescence intensity was completely reversed by the addition of methyl alpha-galactopyranoside. The lectin has two binding sites per molecule for this fluorescent sugar and an association constant of 2.59.10(5) M-1 at 25 degrees C. The binding of N-dansylgalactosamine to the lectin shows that it can accommodate a large hydrophobic substituent on the C-2 carbon of D-galactose. Studies with other sugars indicate that a hydrophobic substituent with alpha-conformation at the anomeric position increases the affinity of binding. The C-4 and C-6 hydroxyl groups are also critical for sugar binding to this lectin.     

Thermodynamics of porphyrin dimerization in aqueous solutions.

The dimerization equilibrium of deuteroporphyrin IX and of mesoporphyrin IX in aqueous solutions were studied by fluorimetric techniques over the 0.01-1 microM concentration range, where dimerization is the dominant aggregation process. Deuteroporphyrin IX was studied at several temperatures over the range 22-37 degrees C, and mesoporphyrin at 25 and 37 degrees C. The magnitudes determined for the dimerization equilibrium constants (25 degrees C, neutral pH, phosphate-buffered saline) are 2.3 X 10(6)M-1 and 5.4 X 10(6)M-1 for the deutero and meso derivatives respectively. The meso, deutero and haemato species tested show a similar temperature effect, namely dimerization decreasing with increasing temperature, indicating the involvement of a negative enthalpy change. Van''t Hoff isochore of the dimerization constants determined for deuteroporphyrin IX was linear within the temperature range of 22-37 degrees C, allowing the calculation of the thermodynamic parameters. For deuteroporphyrin dimerization, those were found to be delta G0 = -36. 4kJ X mol-1; delta H0 = -46. 0kJ X mol-1 and delta S0 = -32.2J X K-1 X mol-1 (at neutral pH, 25 degrees C, phosphate-buffered saline), showing the process to be enthalpy-driven. Similar trends have been found for porphyrin species other than those studied here. Our data fit with a hypothesis giving a major role to the solvent in driving porphyrins to aggregate in aqueous solution. The magnitudes and directions of the energetic changes fit better with the expectation of the '' solvophobic force'' theory predicting enthalpy-driven association, than with the classic hydrophobic bonding, predicting the association to be entropy-driven.     

Binding of branched-chain 2-oxo acids to bovine serum albumin.

1. Binding of branched-chain 2-oxo acids to defatted bovine serum albumin was shown by gel chromatography and equilibrium dialysis. 2. Equilibrium-dialysis data suggest a two-side model for binding in Krebs-Henseleit saline at 37 degrees C with n1 = 1 and n2 = 5. Site association constants were: 4-methyl-2-oxovalerate, k1 = 8.7 x 10(3) M-1, k2 = 0.09 x 10(3) M-1; 3-methyl-2-oxovalerate, k1 = 9.8 x 10(3) M-1, k2 = 0.08 x 10(3) M-1; 3-methyl-2-oxobutyrate, k1 = 1.27 x 10(3) M-1, k2 = less than 0.05 x 10(3) M-1. 3. Binding of 4-methyl-2-oxovalerate to defatted albumin in a phosphate-buffered saline, pH 7.4, gave the following thermodynamic parameters: primary site delta H0(1) = -28.6kJ . mol-1 and delta S0(1) = -15.2J . mol-1 . K-1 (delta G0(1) = -24.0kJ . mol-1 at 37 degrees C) and secondary sites delta H0(2) = -25.4kJ . mol-1 and delta S0(2) = -46.1J . mol-1 . K-1 (delta G0(1) = -11.2kJ . mol-1 at 37 degrees C). Thus binding at both sites is temperature-dependent and increases with decreasing temperature. 4. Inhibition studies suggest that 4-methyl-2-oxovalerate may associate with defatted albumin at a binding site for medium-chain fatty acids. 5. Binding of the 2-oxo acids in bovine, rat and human plasma follows a similar pattern to binding to defatted albumin. The proportion bound in bovine and human plasma is much higher than in rat plasma. 6. Binding to plasma protein, and not active transport, explains the high concentration of branched-chain 2-oxo acids leaving rat skeletal muscle relative to the concentration within the tissue, but does not explain the 2-oxo acid concentration gradient between plasma and liver.     

Binding of 4-methylumbelliferyl beta-D-galactosyl-(1 leads to 3)-N-acetyl-beta-D-galactosaminide to peanut agglutinin. Characterisation and application in substitution titrations

Binding of 4-methylumbelliferyl-2-acetamido-2-deoxy-3-O-(beta-D-galactopyranosyl) beta-D-galactopyranoside [MeUmb beta Gal(beta 1 leads to 3)GalNAc] to peanut agglutinin was characterized by equilibrium dialysis and by measurement of the increase in ultraviolet absorption or fluorescence of the chromophoric glycoside upon continuous titration with excess of the lectin. All data in the 4-30 degrees C range correspond to delta G = -(26.5 +/- 0.1) kJ mol-1, delta H = -(58.4 +/- 2) kJ mol-1 and delta S = -(107 +/- 8)J mol-1 K-1. Values of the association constants are e.g. K = 2.5 X 10(5) M-1 at 4 degrees C and K = 4.5 X 10(4) M-1 at 25 degrees C. MeUmb beta Gal(beta 1 leads to 3)GalNAc was used as an indicator ligand to determine K values for nonchromophoric carbohydrates by continuous displacement titrations, measuring either fluorescence or difference in absorption of the indicator. The data were analyzed in terms of the general expression for a non-ideal indicator system (as detailed in the appendix). Thus, the values of K are not underestimated. They are K = 4.8 X 10(3) M-1 for methyl alpha-D-galactopyranoside [Me alpha Gal], 2.0 X 10(3) M-1 for methyl beta-D-galactopyranoside [Me beta Gal] and 4.7 X 10(3) M-1 for lactose [Gal(beta 1 leads to 4)Glc], all at 14.5 degrees C. The MeUmb difference absorption spectra resulting from binding of the lectin with MeUmb beta Gal(beta 1 leads to 3)GalNAc and MeUmb beta Gal(beta 1 leads to 4)Glc are larger than for MeUmb beta Gal and MeUmb alpha Gal. These observations are consistent with the extended nature of the combining site of peanut agglutinin.     

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.     

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 interaction of a fluorescent DNA oligomer with the anti-tumour drug netropsin.

Fluorescence spectroscopy was used to study the interaction between the minor-groove-binding drug netropsin and the self-complementary oligonucleotide d(CTGAnPTTCAG)2 containing the fluorescent base analogue 2-aminopurine (nP). The binding of netropsin to this oligonucleotide causes strong quenching of the 2-aminopurine fluorescence, observed by steady-state as well as time-resolved spectroscopy. From fluorescence titrations, binding isotherms were recorded and evaluated. The parameters showed one netropsin binding site/oligonucleotide duplex and an association constant of about 10(5) M-1 at 25 degrees C, 3-4 orders of magnitude weaker than for an exclusive adenine/thymine host sequence. From the temperature dependence of the association constant the thermodynamic parameters were obtained as delta G = -29 kJ/mol, delta H = -12 kJ/mol and delta S = +55 J.mol-1.K-1 at 25 degrees C. These parameters resemble those of the interaction of poly[(dG-dC).(dG-dC)] with netropsin, indicating a mainly entropy-driven reaction. The amino group of 2-aminopurine, like that of guanine, resides in the minor groove of DNA. Therefore the relatively weak binding of netropsin to d(CTGAnPTTCAG)2 is probably related to partial blockage of the tight fit of netropsin into the preferred minor groove of an exclusive adenine/thymine host sequence.     

Thermodynamic analysis of ligand binding to winged bean (Psophocarpus tetragonolobus) acidic agglutinin reveals its specificity for terminally monofucosylated H-reactive sugars

The sugar-specific binding of N-dansylgalactosamine to WBA II (n = 2; Ka = 5.6 x 10(3) M-1; delta H = -21 kJ.mol-1; delta S = -21.3 J.mol-1.K-1) was utilized in substitution titrations for evaluating the association constants for the interaction of sugars with the lectin. An axial hydroxyl at C-4 and equatorial hydroxyls at C-3 and C-6 as in D-galacto configuration are crucial for binding. Both axial and equatorial hydroxyls are tolerated at C-2. Conformationally akin disaccharides such as lactose, N-acetyllactosamine, Gal beta 1-3GlcNAc, and Gal beta 1-3GalNAc show similar affinities. 2'-Fucosyllactose and H-disaccharide display 146 and 13 times stronger affinity over lactose and galactose, yet fucose by itself is devoid of activity. An interesting feature, noted for the first time, in protein-sugar interactions is the positive entropy change for the binding of 2'-fucosyllactose, suggesting that nonpolar interactions play an important role in stabilization of the lectin-sugar complex. 3-Fucosyllactose, lactodifucotetraose, lacto-N-fucopentaose II and III are inactive, whereas lacto-N-fucopentaose I has 14-fold lower affinity as compared with 2'-fucosyllactose. Conformational analysis indicates that the substitution at subterminal glucose or GlcNAc by L-fucose in either alpha 1-3 or alpha 1-4 linkage leads to its projection so as to sterically hinder the access of 3'-fucosyllactose, lactodifucotetraose, and lacto-N-fucopentaose II and III to the binding site of winged bean agglutinin II. Similarly the projection of alpha 1-3 linked Gal/GalNAc also leads to steric hindrance and hence prevents the binding of blood group A and B reactive sugars. Considering its unique specificity winged bean agglutinin II should be useful in the isolation and characterization of terminally monofucosylated H-reactive oligosaccharides from those that are difucosylated or internally fucosylated.     

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.     

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.     

Calorimetric studies of the binding of Streptomyces subtilisin inhibitor to subtilisin of Bacillus subtilis strain N'

The binding of Streptomyces subtilisin inhibitor (SSI) to subtilisin of Bacillus subtilis strain N' (subtilisin BPN', EC 3.4.21.14) was studied by isothermal calorimetry at pH 7.0 and at various temperatures ranging from 5 to 30 degrees C. Thermodynamic quantities for the binding reaction were derived as a function of temperature by combining the data reported for the dissociation constant with the present calorimetric results. At 25 degrees C, the values are delta G degrees = -57.9 kJ mol-1, delta H = -19.8 kJ mol-1, delta S degree = 0.13 kJ K-1 mol-1, and delta Cp = -1.02 kJ K-1 mol-1. The entropy and the heat capacity changes are discussed in terms of the contributions from the changes in vibrational modes and in hydrophobic interactions.     

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)     

Microcalorimetric investigation of the interactions in the ternary complex calmodulin-calcium-melittin

Flow microcalorimetric titrations of calmodulin with melittin at 25 degrees C revealed that the formation of the high-affinity one-to-one complex in the presence of Ca2+ (Comte, M., Maulet, Y., and Cox, J. A. (1983) Biochem, J. 209, 269-272) is entirely entropy driven (delta H0 = 30.3 kJ X mol-1; delta S0 = 275 J X K-1 X mol-1). Neither the proton nor the Mg2+ concentrations have any significant effect on the strength of the complex. In the absence of Ca2+, a nonspecific calmodulin-(melittin)n complex is formed; the latter is predominantly entropy driven, accompanied by a significant uptake of protons and fully antagonized by Mg2+. Enthalpy titrations of metal-free calmodulin with Ca2+ in the presence of an equimolar amount of melittin were carried out at pH 7.0 in two buffers of different protonation enthalpy. The enthalpy and proton release profiles indicate that: protons, absorbed by the nonspecific calmodulin-melittin complex, are released upon binding of the first Ca2+; Ca2+ binding to the high-affinity configuration of the calmodulin-melittin complex displays an affinity constant greater than or equal to 10(7) M-1, i.e. 2 orders of magnitude higher than that of free calmodulin; the latter is even more entropy driven (delta H0 = 7.2 kJ X site-1; delta S0 = 158 J X K-1 X site-1) than binding to free calmodulin (delta H0 = 4.7 kJ X site-1; delta S0 = 112 J X K-1 X site-1), thus underlining the importance of hydrophobic forces in the free energy coupling involved in the ternary complex.     

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.     

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.     

Thermodynamics of DNA hairpins: contribution of loop size to hairpin stability and ethidium binding.

A combination of calorimetric and spectroscopic techniques was used to evaluate the thermodynamic behavior of a set of DNA hairpins with the sequence d(GCGCTnGCGC), where n = 3, 5 and 7, and the interaction of each hairpin with ethidium. All three hairpins melt in two-state monomolecular transitions, with tm's ranging from 79.1 degrees C (T3) to 57.5 degrees C (T7), and transition enthalpies of approximately 38.5 kcal mol-1. Standard thermodynamic profiles at 20 degrees C reveal that the lower stability of the T5 and T7 hairpins corresponds to a delta G degree term of +0.5 kcal mol-1 per thymine residue, due to the entropic ordering of the thymine loops and uptake of counterions. Deconvolution of the ethidium-hairpin calorimetric titration curves indicate two sets of binding sites that correspond to one ligand in the stem with binding affinity, Kb, of approximately 1.8 x 10(6) M-1, and two ligands in the loops with Kb of approximately 4.3 x 10(4) M-1. However, the binding enthalpy, delta Hb, ranges from -8.6 (T3) to -11.6 kcal mol-1 (T7) for the stem site, and -6.6 (T3) to -12.7 kcal mol-1 (T7) for the loop site. Relative to the T3 hairpin, we obtained an overall thermodynamic contribution (per dT residue) of delta delta Hb = delta(T delta Sb) = -0.7(5) kcal mol-1 for the stem sites and delta delta Hb = delta(T delta Sb) = -1.5 kcal mol-1 for the loop sites. Therefore, the induced structural perturbations of ethidium binding results in a differential compensation of favorable stacking interactions with the unfavorable ordering of the ligands.     

Microcalorimetric investigation of the interaction of calmodulin with seminalplasmin and myosin light chain kinase

Flow microcalorimetric titrations of calmodulin with seminalplasmin at 25 degrees C revealed that the high affinity one-to-one complex in the presence of Ca2+ (Comte, M., Malnoe, A., and Cox, J. A. (1986) Biochem. J. 240, 567-573) is entirely enthalpy-driven (delta H0 = -50 kJ.mol-1; delta S0 = O J.K-1.mol-1; delta Cp0 = O J.K-1.mol-1) and is not influenced by the proton or Mg2+ concentration. The Sr2+- and Cd2+-promoted high affinity complexes are also exothermic for -49 and -45 kJ.mol-1, respectively. The observed low affinity interaction in the absence of divalent ions displays no enthalpy change. No enthalpy changes are observed when calmodulin and seminalplasmin are mixed in the presence of millimolar concentrations of Mg2+, Zn2+, or Mn2+. Enthalpy titrations of the 1:1 calmodulin-seminalplasmin complex with Ca2+ and of partly Ca2+-saturated calmodulin with seminalplasmin revealed that only the species calmodulin.Can greater than or equal to 2 is fully competent for high affinity interaction with seminalplasmin. Binding of the second Ca2+ is strongly enhanced (K2 greater than or equal to 5 X 10(7) M-1) as compared to that in free calmodulin (K2 = 2.6 X 10(5) M-1). This is essentially due to the concomitant strongly exothermic step of isomerization of the calmodulin-seminalplasmin complex from its low to its high affinity form. Binding of the remaining two Ca2+ to the high affinity seminalplasmin-calmodulin complex displays the same affinity constants and endothermic enthalpy change as in free calmodulin. A microcalorimetric study on the complex formation between Ca2+-saturated calmodulin and turkey gizzard myosin light chain kinase revealed that the interaction is strongly exothermic with an important overall gain of order (delta H0 = -85 kJ.mol-1; delta S0 = -122 J.K-1.mol-1) and occurs with significant proton uptake (0.44 H+ per mol at pH 7.5). The observed low affinity interaction (K = 2.2 X 10(5) M-1) in the absence of Ca2+ (Mamar-Bachi, A., and Cox, J. A. (1987) Cell Calcium 8, 473-482) displays neither a change in enthalpy nor in protonation.