DeltaCp Degrees Research Articles

Energetic diversity of DNA minor-groove recognition by small molecules displayed through some model ligand-DNA systems.

Energetics of interactions occurring in the model ligand-DNA systems constituted from distamycin A (DST), netropsin (NET) and the oligomeric duplexes d(GCAAGTTGCGATATACG)d(CGTATATCGCAACTTGC)=D#1 and d(GCAAGTTGCGAAAAACG)d(CGTTTTTCGCAACTTGC)=D#2 was studied by spectropolarimetry, UV-absorption spectroscopy and isothermal titration calorimetry. Model analysis of the measured signals was applied to describe individual and competitive binding in terms of populations of various species in the solution. Our results reveal several unprecedented ligand-DNA binding features. DST binds to the neighboring 5'-AAGTT-3' and 5'-ATATA-3' sites of D#1 statistically in a 2:1 binding mode. By contrast, its association to D#2 appears to be a 2:1 binding event only at the DST/D#2 molar ratios between 0 and 2 while its further binding to D#2 may be considered as a step-by-step binding to the unoccupied 5'-AAAAA-3' sites resulting first in DST3D#2 and finally in DST4D#2 complex formation. Competition between DST and NET binding shows that for the most part DST displaces NET from its complexes with D#1 and D#2. In contrast to the obligatory 1:1 binding of DST to the ligand-free 5'-AAAAA-3' sites observed at DST/5'-AAAAA-3' <1 the displacement of NET bound to the 5'-AAAAA-3' sites by added DST occurs even at the smallest additions of DST in a 2:1 manner. NET can also displace DST molecules but only those bound monomerically to the 5'-AAAAA-3' sites of DST3D#2. Actually, only half of these molecules can be displaced due to the simultaneous rebinding of the displaced DST to the unreacted 5'-AAAAA-3' sites in DST3D#2. Binding of DST and NET to D#1 and D#2 is an enthalpy driven process accompanied by large unfavorable (DST), small (NET) or large favorable (NET binding to 5'-AAAAA-3') entropy contributions and negative deltaCP degrees that are reasonably close to deltaCP degrees predicted from the calculated changes in solvent-accessible surface areas that accompany complex formation. Although various modes of DST and NET binding within D#1 and D#2 are characterized by significant energetic differences they seem to be governed by the same driving forces; the hydrophobic transfer of ligand from the solution into the duplex binding site and the accompanying specific non-covalent ligand-DNA and ligand-ligand interactions occurring within the DNA minor groove.

Thermodynamic analysis of the binding of glutathione to glutathioneS-transferase over a range of temperatures

The binding properties of a glutathione S-transferase (EC 2.5.1.18) from Schistosoma japonicum to substrate glutathione (GSH) has been investigated by intrinsic fluorescence and isothermal titration calorimetry (ITC) at pH 6.5 over a temperature range of 15-30 degrees C. Calorimetric measurements in various buffer systems with different ionization heats suggest that protons are released during the binding of GSH at pH 6.5. We have also studied the effect of pH on the thermodynamics of GSH-GST interaction. The behaviour shown at different pHs indicates that at least three groups must participate in the exchange of protons. Fluorimetric and calorimetric measurements indicate that GSH binds to two sites in the dimer of 26-kDa glutathione S-transferase from Schistosoma japonicum (SjGST). On the other hand, noncooperativity for substrate binding to SjGST was detected over a temperature range of 15-30 degrees C. Among thermodynamic parameters, whereas DeltaG degrees remains practically invariant as a function of temperature, DeltaH and DeltaS degrees both decrease with an increase in temperature. While the binding is enthalpically favorable at all temperatures studied, at temperatures below 25 degrees C, DeltaG degrees is also favoured by entropic contributions. As the temperature increases, the entropic contributions progressively decrease, attaining a value of zero at 24.3 degrees C, and then becoming unfavorable. During this transition, the enthalpic contributions become progressively favorable, resulting in an enthalpy-entropy compensation. The temperature dependence of the enthalpy change yields the heat capacity change (DeltaCp degrees ) of -0.238 +/- 0.04 kcal per K per mol of GSH bound.

A Thermodynamic Molecular Switch in Biological Systems: Ribonuclease S' Fragment Complementation Reactions

Conference Abstract| October 01 2000 A Thermodynamic Molecular Switch in Biological Systems: Ribonuclease S' Fragment Complementation Reactions P. W. Chun P. W. Chun 1Box 100245 Gainesville FL 32610–0245 Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (2000) 28 (5): A410. https://doi.org/10.1042/bst028a410b Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Twitter LinkedIn Cite Icon Cite Get Permissions Citation P. W. Chun; A Thermodynamic Molecular Switch in Biological Systems: Ribonuclease S' Fragment Complementation Reactions. Biochem Soc Trans 1 October 2000; 28 (5): A410. doi: https://doi.org/10.1042/bst028a410b Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu nav search search input Search input auto suggest search filter All ContentAll JournalsBiochemical Society Transactions Search Advanced Search This content is only available as a PDF. © 2000 Biochemical Society2000 Article PDF first page preview Close Modal You do not currently have access to this content.

Calorimetric examination of high-affinity Src SH2 domain-tyrosyl phosphopeptide binding: dissection of the phosphopeptide sequence specificity and coupling energetics.

SH2 domains are protein modules which interact with specific tyrosine phosphorylated sequences in target proteins. The SH2 domain of the Src kinase binds with high affinity to a tyrosine phosphorylated peptide containing the amino acids Glu, Glu, and Ile (EEI) at the positions +1, +2, and +3 C-terminal to the phosphotyrosine, respectively. To investigate the degree of selectivity of the Src SH2 domain for each amino acid of the EEI motif, the binding thermodynamics of a panel of substitutions at the +1 (Gln, Asp, Ala, Gly), +2 (Gln, Asp, Ala, Gly), and +3 (Leu, Val, Ala, Gly) positions were examined using titration microcalorimetry. It was revealed that the Src SH2 domain is insensitive (DeltaDeltaG degrees </= 0.6 kcal/mol) to conservative substitutions at all three peptide positions. However, mutation to Ala resulted in moderate reductions in DeltaG degrees, with the substitution at the +3 position showing the largest loss in affinity (DeltaDeltaG degrees = 1.4 kcal/mol), followed by the +2 (DeltaDeltaG degrees = 1.0 kcal/mol) and +1 (DeltaDeltaG degrees = 0.5 kcal/mol) positions. This hierarchy of binding was not reflected in the values of the heat capacity change, since only the peptide substituted to Ala at the +3 position showed a DeltaCp degrees that was reduced in magnitude compared to wild-type. To assess the degree of cooperation upon binding (or coupling) between the amino acids of the EEI sequence, the binding of a series of singly, doubly, and triply Ala substituted phosphopeptides was examined and analyzed using double mutant cycles. It was revealed that the effects of the Ala substitutions on DeltaG degrees were additive. However, nonadditive binding enthalpies were observed between the +1 Glu and +3 Ile, as well as the +2 Glu and +3 Ile, suggesting that communication occurs between residues of the EEI motif upon binding.

Probing the "two-pronged plug two-holed socket" model for the mechanism of binding of the Src SH2 domain to phosphotyrosyl peptides: a thermodynamic study.

Src homology 2 (SH2) domains are protein modules that specifically bind to tyrosyl phosphorylated peptides on signaling proteins. X-ray crystallographic studies of the SH2 domain of the Src kinase have probed the mechanism of binding, leading to the "two-pronged plug two-holed socket" mechanism whereby binding is hypothesized to resemble a two-pronged plug (the peptide) inserting into a two-holed socket (the SH2 domain). This binding model predicts (1) a hydrophobic basis for high-affinity binding largely determined by the level of insertion of the third residue C-terminal to the phosphotyrosine in the peptide into a primarily hydrophobic pocket (the +3 binding pocket) of the SH2 domain, and (2) a binding mechanism involving no significant conformational changes in the SH2 domain. In this study, we have probed these predictions by using isothermal titration calorimetry to extract complete thermodynamic profiles (Delta G degrees, Delta H degrees, Delta S degrees, Delta Cp degrees) for the binding of the Src SH2 domain to two series of tyrosyl phosphopeptides. One series consisted of peptides that have been determined by X-ray crystallography to have different levels of insertion of the peptide's +3 position into the +3 binding pocket. The other series consisted of peptides with progressively smaller hydrophobic side chains (I, L, V, and A) at the +3 position. Consistent with a binding mechanism that does not involve substantial conformational changes, the Delta Cp degrees values for all peptides were small and, at least for the high-affinity interactions, similar to the Delta Cp degrees values predicted from surface area calculations. However, unexpectedly, this study reveals that high-affinity binding was only partially determined by the interactions between the +3 residue in the peptide and the +3 binding pocket. Furthermore, the Delta Cp degrees values for all peptides studied were similar, implying similar degrees of desolvation of the +3 binding pocket upon binding. These results indicate that the "two-pronged plug two-holed socket" model is an oversimplification of the Src SH2 domain binding mechanism.

Thermodynamics of the interaction of human immunoglobulin E with its high-affinity receptor Fc epsilon RI.

We have employed isothermal titration calorimetry (ITC) and circular dichroism (CD) spectroscopy to characterize the binding of soluble fragments of IgE (IgE-Fc and Fc epsilon 3-4) to a soluble fragment of the high-affinity receptor Fc epsilon RI alpha-chain (sFc epsilon RI alpha). The thermodynamic parameters for the interaction of IgE-Fc and Fc epsilon 3-4 with sFc epsilon RI alpha, determined using ITC, confirm the earlier conclusion that the C epsilon 2 domain is not involved in the interaction and that the stoichiometry of both complexes is 1:1. For both IgE-Fc and Fc epsilon 3-4, the value of Delta H degrees is -36.9 +/- 4.6 kcal mol-1 at 37.3 degreesC and Delta Cp degrees is -820 +/- 120 cal mol-1 K-1. The temperature at which DeltaS degrees is zero is 284 +/- 1 K, indicating that the entropy contribution to the thermodynamics of association is unfavorable at physiological temperature. Of particular interest is the large value of Delta Cp degrees. The large surface area of IgE and Fc epsilon RI alpha that is implicated in complex formation from previous mutagenesis studies on the two proteins may account in part for the magnitude of Delta Cp degrees. Additional contributions may arise from hydration within the binding site and changes in tertiary structure of the individual components of the complex. However, the CD spectra of IgE, IgE-Fc, and Fc epsilon 3-4 complexes with sFc epsilon RI alpha are merely the sum of the spectra of their individual components, indicating that the secondary structure of the immunoglobulin domain folds are preserved on complex formation. Thus, any change in tertiary structure must be limited to the relative disposition of the immunoglobulin domains C epsilon 3 and C epsilon 4 in IgE and the two immunoglobulin-like domains in the alpha-chain of Fc epsilon RI.

Temperature dependency of thermodynamic parameters in interactions between hen egg-white lysozyme (HEL) and anti-HEL antibodies.

We examined temperature dependency of thermodynamic parameters in the interactions between hen egg-white lysozyme (HEL) and anti-HEL antibody, D1.3, and two mutant antibodies. The DeltaH degrees values appeared to decrease biphasically in the temperature range from 10 to 45 degrees C with the apparent inflection point around 30 degrees C. The DeltaG degrees calculated from the KA values showed only small differences because of entropy and enthalpy compensation. It has been argued that large negative values of heat capacity change (DeltaCp degrees), if observed, are mainly derived from hydrophobic interactions. However, the observed DeltaCp degrees values were too high to be ascribed only to hydrophobic interactions. Moreover, addition of methanol did not cause a decrease in the absolute value of DeltaCp degrees.

Energetics of two-step binding of a chromophoric reaction product, trans-3-indoleacryloyl-CoA, to medium-chain acyl-coenzyme-A dehydrogenase.

We previously demonstrated that the UV/visible spectrum of a chromophoric ligand, trans-3-indoleacryloyl-coenzyme-A (IACoA), is red-shifted (due to polarization of its carbonyl group) upon binding to pig kidney medium-chain acyl-CoA dehydrogenase (MCAD). The transient kinetic data revealed that the overall binding occurred in two steps. The first (fast) step involved the formation of an MCAD-IACoA collision complex in which the electronic structure of IACoA remained unchanged (the "colorless" complex), followed by a slow isomerization step with a concomitant red-shift in the IACoA spectrum (the "colored" complex) [Johnson, J. K., Wang, Z. X., and Srivastava, D. K. (1992) Biochemistry 31, 10564-10575]. To ascertain the energetics of the above two-step process, we investigated the temperature dependence of the spectral changes, binding constant of the MCAD-IACoA complex, and the rate constants for the conversion between the colorless and colored complexes. The data revealed that as the temperature of the incubation mixture of MCAD and IACoA ([IACoA] >> [MCAD] > Kd) increases from 12 to 35 degrees C, the resultant spectral peak of the MCAD-IACoA complex (lambda max = 417 nm) decreases. However, in this temperature range, the equilibrium constant for the second (isomerization) step remains unaffected. Isothermal titration calorimetric studies for the binding of IACoA to MCAD reveal that the overall binding energy at 25 degrees C (delta G degree = -7.4 kcal/mol) is contributed almost equally by the enthalpic (delta H degree = -3.7 kcal/mol) and entropic (T delta S degree = 3.7 kcal/mol) changes. As the temperature increases, both delta H degree and T delta S degree decrease proportionately, resulting in a strong enthalpy-entropy compensation effect. The temperature dependence of delta H degree yields a delta Cp degree value of -0.24 kcal/mol. The data presented herein throw light on the energetic consequences for the binding of IACoA to MCAD, the apparent similarity between the van't Hoff and calorimetric enthalpies, enthalpic and entropic contributions during the polarization of the carbonyl group of IACoA, and the overall structural-functional features of the enzyme-ligand complex as well as enzyme catalysis.

The effects of temperature on the interactions between volatile general anaesthetics and a neuronal nicotinic acetylcholine receptor.

1. Completely isolated identified neurones from the right parietal ganglion of the pond snail Lymnaea stagnalis were investigated under two-electrode voltage clamp. Neuronal nicotinic acetylcholine receptor (AChR) currents were studied at low acetylcholine concentrations (< or = 200 nM). 2. Inhibition of the ACh-induced currents by three volatile general anaesthetics (halothane, isoflurane and methoxyflurane) and the specific inhibitor (+)-tubocurarine was studied as a function of temperature (over the range 4-25 degrees C). 3. The inhibition by the volatile anaesthetics increased (inhibition constants decreased) with decreasing temperature while the inhibition by (+)-tubocurarine did not change significantly near room temperature, but decreased at lower temperatures. The (+)-tubocurarine inhibition appeared to be competitive in nature and showed no significant voltage-dependence. 4. The van't Hoff plots (logarithms of the dissociation constants against reciprocal absolute temperature) were linear for the anaesthetics, but markedly non-linear for (+)-tubocurarine. From these plots, values for the changes in the standard Gibbs free energy delta G degrees water-->AChR, enthalpy delta H degree water-->AChR, entropy delta S degree water-->AChR and heat capacity delta Cp degree water-->AChR were determined. Tubocurarine was found to bind very much tighter to the receptor than the volatile anaesthetics due, entirely, to a favourable increase in entropy on binding. 5. A comparison between the temperature-dependence of the anaesthetic inhibition of the ACh receptor and that of general anaesthetic potencies in animals indicates that the temperature-dependence of animal potencies might be simply accounted for in terms of changes in anaesthetic/receptor binding.

Thermodynamic and Kinetic Characterization of the Binding of the TATA Binding Protein to the Adenovirus E4 Promoter

A thermodynamic analysis of the binding of the TATA binding protein (TBP) from Saccharomyces cerevisiae to the adenovirus E4 promoter was conducted using quantitative DNase I "footprint" titration techniques. These studies were conducted to provide a foundation for studies of TBP structure-function relations and its assembly into transcription preinitiation complexes. The binding of TBP to the E4 promoter is well described by the Langmuir binding polynomial, suggesting that no linked equilibria contribute to the binding reaction under the conditions examined. Van't Hoff analysis yielded a nonlinear dependence on temperature with the TBP-E4 promoter interaction displaying maximal affinity at 30 degrees C. An unusually negative value of the apparent standard heat capacity change, delta Cp degrees = -3.5 +/- 0.5 kcal/mol.K, was determined from these data. The dependence of the TBP-E4 promoter interaction on [KCl] indicates that 3.6 +/- 0.3 K+ ions are displaced upon complex formation. Within experimental error, no linkage of proton binding with the TBP-E4 promoter interaction is detectable between pH 5.9 and 8.7. Rates of association of TBP for the E4 promoter were obtained using a novel implementation of a quench-flow device and DNase I "footprinting" techniques. The value determined for the second-order rate constant at pH 7.4, 100 mM KCl, 5 mM MgCl2, 1 mM CaCl2, 30 degrees C (ka = 5.2 +/- 0.5) x 10(5) M-1 s-1) confirms the results obtained by Hawley and co-workers [Hoopes, B.C., LeBlanc, J.F., & Hawley, D.K. (1992) J. Biol. Chem. 267, 11539-11547] and extends them through TBP concentrations of 636 nM.(ABSTRACT TRUNCATED AT 250 WORDS)

Thermodynamics of ligand binding to trp repressor.

The thermodynamics of L-tryptophan and operator DNA binding to the tryptophan repressor of Escherichia coli were analyzed by titration microcalorimetry and van't Hoff analysis of footprinting titrations, respectively. At 25 degrees C in 10 mM sodium phosphate, pH 7.6, and 0.1 M NaCl, the binding of L-tryptophan to the repressor is characterized by values of delta G degrees = -6.04, delta H degree = -14.7, and T delta S degree = -8.67 kcal/mol. The temperature dependence of delta H degree yields delta Cp degree = -0.46 +/- 0.08 kcal/(mol.K) per dimer. The binding is noncooperative at all temperatures studied. At 23 degrees C in 2.5 mM sodium phosphate, pH 7.6, and 25 mM NaCl, the binding of operator DNA to the repressor is characterized by values of delta G degree = -13.3 kcal/mol, delta H degree = -1.55 kcal/mol, T delta S degree = 11.8 kcal/mol, and delta Cp degree = -0.54 +/- 0.10 kcal/(mol.K). Changes in water-accessible surface areas upon binding of L-tryptophan or DNA were calculated from X-ray crystal structures. The experimentally observed delta Cp degree values were compared with delta Cp degree values calculated according to several methods based on various proposed relationships between surface area changes and heat capacity changes. Regardless of which method is used, we find poor agreement between the calorimetric results for L-tryptophan binding and the surface areas calculated from X-ray data; the direction of the discrepancy is that the X-ray data underestimate the value of delta Cp degree.(ABSTRACT TRUNCATED AT 250 WORDS)

Thermodynamics of the glucocorticoid receptor-DNA interaction: Binding of wild-type GR DBD to different response elements

We used fluorescence spectroscopy to study the chemical equilibria between an 82-residue protein fragment containing the core conserved region of the glucocorticoid receptor DNA-binding domain (GR DBD) and a palindromic glucocorticoid response element (GRE), a consensus GRE half-site, a consensus estrogen response element (ERE) half-site, and two intermediate half-sites (GRE2 and ERE2). Equilibrium parameters were determined at 20 degrees C and buffer conditions that approximate intracellular conditions. The association constants for GR DBD binding to the GRE (5'TGTTCT3') and GRE2 (5'TGTCCT3') half-sites at 85 mM NaCl, 100 mM KCl, 2 mM MgCl2, and 20 mM Tris-HCl at pH 7.4 and low concentrations of an antioxidant and a nonionic detergent are (1.0 +/- 0.1) x 10(6) M-1 and (5.1 +/- 0.2) x 10(5) M-1, respectively. The association constants for binding to the ERE (5'TGACCT3') and ERE2 (5'TGATCT3') half-sites are < 10(5) M-1. The implications of these numbers for the specificity and affinity for the binding of the intact GR to DNA are discussed. Comparison of GR DBD binding to a GRE half-site and a palindromic GRE sequence allowed us to estimate the cooperativity parameter, omega obs = 25-50, for GR DBD binding to GRE. The thermodynamics of the GR DBD interaction with a GRE half-site were also investigated by determining the temperature dependence of the observed association constant. The nonlinear dependence in ln Kobs as a function of 1/T is consistent with a change in standard heat capacity, delta Cp degree obs = 1.0 +/- 0.2 kcal mol-1 K-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Stability of recombinant Lys25-ribonuclease T1

The conformational stability of recombinant Lys25-ribonuclease T1 has been determined by differential scanning microcalorimetry (DSC), UV-monitored thermal denaturation measurements, and isothermal Gdn.HCl unfolding studies. Although rather different extrapolation procedures are involved in calculating the Gibbs free energy of stabilization, there is fair agreement between the delta G degrees values derived from the three different experimental techniques at pH 5, theta = 25 degrees C: DSC, 46.6 +/- 2.1 kJ/mol; UV melting curves, 48.7 +/- 5 kJ/mol; Gdn.HCl transition curves, 40.8 +/- 1.5 kJ/mol. Thermal unfolding of the enzyme is a reversible process, and the ratio of the van't Hoff and calorimetric enthalpy, delta HvH/delta Hcal, is 0.97 +/- 0.06. This result strongly suggests that the unfolding equilibrium of Lys25-ribonuclease T1 is adequately described by a simple two-state model. Upon unfolding the heat capacity increases by delta Cp degrees = 5.1 +/- 0.5 kJ/(mol.K). Similar values have been found for the unfolding of other small proteins. Surprisingly, this denaturational heat capacity change practically vanishes in the presence of moderate NaCl concentrations. The molecular origin of this effect is not clear; it is not observed to the same extent in the unfolding of bovine pancreatic ribonuclease A, which was employed in control experiments. NaCl stabilizes Lys25-ribonuclease T1. The transition temperature varies with NaCl activity in a manner that suggests two limiting binding equilibria to be operative. Below approximately 0.2 M NaCl activity unfolding is associated with dissociation of about one ion, whereas above that concentration about four ions are released in the unfolding reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Thermodynamics of industrially-important, enzyme-catalyzed reactions

The thermodynamics of 10 industrially-important, enzyme-catalyzed reactions are examined. The reactions discussed are: the conversions of penicillin G to 6-amino-penicillinic acid using the enzyme penicillin acylase; starch to glucose using amylases; glucose to fructose using glucose (xylose) isomerase; cellulose to glucose using cellulase; fumaric acid and ammonia to L-aspartic acid using L-aspartase; transcinnamic acid and ammonia to L-phenylalanine using L-phenylalanine ammonia lyase; L-histidine to urocanic acid and ammonia using L-histidine ammonia lyase; lactose to glucose and galactose using lactase; and the reactions catalyzed by amino acylases and proteases. The selection of these processes was based on the economic value of the products and their intrinsic industrial importance. The available thermodynamic properties, such as equilibrium constants, Gibbs energies (delta G degrees), enthalphies (delta H degrees), and heat capacity changes (delta Cp degrees) of these enzyme-catalyzed reactions, are reviewed and summarized. Recommendations are made for future research in this area.

Hydrophobic effect in protein folding and other noncovalent processes involving proteins.

Large negative standard heat capacity changes (delta CP degree much less than 0) are the hallmark of processes that remove nonpolar surface from water, including the transfer of nonpolar solutes from water to a nonaqueous phase and the folding, aggregation/association, and ligand-binding reactions of proteins [Sturtevant, J. M. (1977) Proc. Natl. Acad. Sci. USA 74, 2236-2240]. More recently, Baldwin [Baldwin, R. L. (1986) Proc. Natl. Acad. Sci. USA 83, 8069-8072] proposed that the delta CP degree of protein folding could be used to quantify the contribution of the burial of nonpolar surface (the hydrophobic effect) to the stability of a globular protein. We demonstrate that identical correlations between the delta CP degree and the change in water-accessible nonpolar surface area (delta Anp) are obtained for both the transfer of nonpolar solutes from water to the pure liquid phase and the folding of small globular proteins: delta CP degree/delta Anp = -(0.28 +/- 0.05) (where delta Anp is expressed in A2 and delta CP degree is expressed in cal.mol-1.K-1; 1 cal = 4.184 J). The fact that these correlations are identical validates the proposals by both Sturtevant and Baldwin that the hydrophobic effect is in general the dominant contributor to delta CP degree and provides a straightforward means of estimating the contribution of the hydrophobic driving force (delta Ghyd degree) to the standard free energy change of a noncovalent process characterized by a large negative delta CP degree in the physiological temperature range: delta Ghyd degree congruent to (80 +/- 10)delta CP degree.

Thermodynamics of concanavalin A dimer-tetramer self-association: sedimentation equilibrium studies.

The effects of temperature and pH on the reversible dimer-tetramer association of concanavalin A were studied by the high-speed sedimentation equilibrium technique. Both commercial and highly purified preparations of concanavalin A were used. Equilibrium constants were analyzed as a Wyman linked function by using truncated van't Hoff temperature dependence. In the concentration range 0.1-3.0 mg/mL, from 5 to 35 degrees C and between pH 5.5 and pH 7.5 at 0.5 M ionic strength, only dimer and tetramer species were present in both preparations. For purified concanavalin A, association constants ranged from 1.5 X 10(3) to 8.0 X 10(7) M-1. Constants for our commercial preparation were approximately 10-fold lower due to the decrease competency of some subunits to self-associate. From the fit of the Wyman model to the experimental data, delta G degree, delta H degree, delta S degree, and delta Cp degree were calculated for the association and association-linked ionization reactions. From the values of the ionization thermodynamic parameters, the association is governed by the ionization of a histidine side chain on each subunit, either histidine-51 or histidine-121. The association is characterized by large entropy (66.3 cal.mol-1.deg-1 at 25 degrees C) and heat capacity (-821 cal.mol-1.deg-1) changes in accordance with the large hydrophobic association surface observed in crystallographic studies [Reeke, G. N., Jr., Becker, J. W., & Edelman, G. M. (1975) J. Biol. Chem. 250, 1525-1547]. In addition, there is a large enthalpy change (10.4 kcal.mol-1 at 25 degrees C). We propose a model for the interaction based on a more detailed thermodynamic description than was obtained in an earlier, incomplete study [Huet, M., & Clavarie, J. M. (1978) Biochemistry 17, 236-241].