Studies of the pH dependence of the formation of binary and ternary complexes with liver alcohol dehydrogenase

The association of the coenzyme NAD+ to liver alcohol dehydrogenase (LADH) is known to be pH dependent, with the binding being linked to the shift in the pK of some group on the protein from a value of 9-10, in the free enzyme, to 7.5-8 in the LADH-NAD+ binary complex. We have further characterized the nature of this linkage between NAD+ binding and proton dissociation by studying the pH dependence (pH range 6-10) of the proton release, delta n, and enthalpy change, delta Ho(app), for formation of both binary (LADH-NAD+) and ternary (LADH-NAD+-I, where I is pyrazole or trifluoroethanol) complexes. The pH dependence of both delta n and delta Ho(app) is found to be consistent with linkage to a single acid dissociating group, whose pK is perturbed from 9.5 to 8.0 upon NAD+ binding and is further perturbed to approximately 6.0 upon ternary complex formation. The apparent enthalpy change for NAD+ binding is endothermic between pH 7 and pH 10, with a maximum at pH 8.5-9.0. The pH dependence of the delta Ho(app) for both binary and ternary complex formation is consistent with a heat of protonation of -7.5 kcal/mol for the coupled acid dissociating group. The intrinsic enthalpy changes for NAD+ binding and NAD+ plus pyrazole binding to LADH are determined to be approximately 0 and -11.0 kcal/mol, respectively. Enthalpy change data are also presented for the binding of the NAD+ analogues adenosine 5'-diphosphoribose and 3-acetylpyridine adenine dinucleotide.     

Two-dimensional differential scanning calorimetry: simultaneous resolution of intrinsic protein structural energetics and ligand binding interactions by global linkage analysis.

A general theoretical development for the design and analysis of two-dimensional thermal stability surfaces of proteins is presented. The surfaces are generated from multiple excess heat capacity profiles ( vs T) obtained at varying concentrations of an interacting ligand. The energetics of both the intrinsic protein stability and the protein-ligand interaction are simultaneously resolved by employing statistical thermodynamic models in global linkage analysis. This formalism allows resolution of the intrinsic protein folding-unfolding parameters (enthalpy, entropy, and heat capacity changes) as well as the ligand interaction parameters (binding stoichiometry, enthalpy, entropy, and heat capacity changes). The theory has been applied to the case of ribonuclease A and its interaction with cytidine-2'-monophosphate. The accuracy of the thermodynamic parameters obtained by this approach compares within error with those parameters that can be obtained by direct measurements.     

Energetics of solvent and ligand-induced conformational changes in alpha-lactalbumin

The energetics of structural changes in the holo and apo forms of a-lactalbumin and the transition between their native and denatured states induced by binding Ca2+ and Na+ have been studied by differential scanning and isothermal titration microcalorimetry and circular dichroism spectroscopy under various solvent conditions. Removal of Ca2+ from the protein enhances its sensitivity to pH and ionic conditions due to noncompensated negative charge-charge interactions at the cation binding site, which significantly reduces its overall stability. At neutral pH and low ionic strength, the native structure of apo-alpha-lactalbumin is stable below 14 C and undergoes a conformational change to a native-like molten globule intermediate at temperatures above 25 degrees C. The denaturation of either holo- or apo-alpha-lactalbumin is a highly cooperative process that is characterized by an enthalpy of similar magnitude when calculated at the same temperature. Measured by direct calorimetric titration, the enthalpy of Ca2+-binding to apo-LA at pH 7.5 is -7.1 kJ mol(-1) at 5.0 degrees C. which is essentially invariant to protonation effects. This small enthalpy effect infers that stabilization of alpha-lactalbumin by Ca2+ is primarily an entropy driven process, presumably arising from electrostatic interactions and the hydration effect. In contrast to the binding of calcium, the interaction of sodium with apo-LA does not produce a noticeable heat effect and is characterized by its ionic nature rather than specific binding to the metal-binding site. Characterization of the conformational stability and ligand binding energetics of alpha-lactalbumin as a function of solvent conditions furnishes significant insight regarding the molecular flexibility and regulatory mechanism mediated by this protein.     

Binding-linked protonation of a DNA minor-groove agent

The energetics for binding of a diphenyl diamidine antitrypanosomal agent CGP 40215A to DNA have been studied by spectroscopy, isothermal titration calorimetry, and surface plasmon resonance biosensor methods. Both amidines are positively charged under experimental conditions, but the linking group for the two phenyl amidines has a pK(a) of 6.3 that is susceptible to a protonation process. Spectroscopic studies indicate an increase of 2.7 pK(a) units in the linking group when the compound binds to an A/T minor-groove site. Calorimetric titrations in different buffers and pH conditions support the proton-linkage process and are in a good agreement with spectroscopic titrations. The two methods established a proton-uptake profile as a function of pH. The exothermic enthalpy of complex formation varies with different pH conditions. The observed binding enthalpy increases as a function of temperature indicating a negative heat capacity change that is typical for DNA minor-groove binders. Solvent accessible surface area calculations suggest that surface burial accounts for about one-half of the observed intrinsic negative heat capacity change. Biosensor and calorimetric experiments indicate that the binding affinities vary with pH values and salt concentrations due to protonation and electrostatic interactions. The surface plasmon resonance binding studies indicate that the charge density per phosphate in DNA hairpins is smaller than that in polymers. Energetic contributions from different factors were also estimated for the ligand/DNA complex.     

Intrinsic affinity of protein – ligand binding by differential scanning calorimetry

Differential scanning calorimetry (DSC) determines the enthalpy change upon protein unfolding and the melting temperature of the protein. Performing DSC of a protein in the presence of increasing concentrations of specifically-binding ligand yields a series of curves that can be fit to obtain the protein–ligand dissociation constant as done in the fluorescence-based thermal shift assay (FTSA, ThermoFluor, DSF). The enthalpy of unfolding, as directly determined by DSC, helps improving the precision of the fit. If the ligand binding is linked to protonation reactions, the intrinsic binding constant can be determined by performing the affinity determination at a series of pH values. Here, the intrinsic, pH-independent, affinity of acetazolamide binding to carbonic anhydrase (CA) II was determined. A series of high-affinity ligands binding to CAIX, an anticancer drug target, and CAII showed recognition and selectivity for the anticancer isozyme. Performing the DSC experiment in buffers of highly different enthalpies of protonation enabled to observe the ligand unbinding-linked protonation reactions and estimate the intrinsic enthalpy of binding. The heat capacity of combined unfolding and unbinding was determined by varying the ligand concentrations. Taken together, these parameters provided a detailed thermodynamic picture of the linked ligand binding and protein unfolding process.     

Thermodynamic analysis of binding and protonation in DOTAP/DOPE (1:1): DNA complexes using isothermal titration calorimetry

A better understanding of the nature of the interaction between various cationic lipids used for gene delivery and DNA would lend insight into their structural and physical properties that may modulate their efficacy. We therefore separated the protonation and binding events which occur upon complexation of 1:1 DOTAP (1,2-dioleoyl-3-trimethylammonium propane):DOPE (1,2-dioleoylphosphatidylethanolamine) liposomes to DNA using proton linkage theory and isothermal titration calorimetry (ITC). The enthalpy of DOPE protonation was estimated as -45.0+/-0.7 kJ/mol and the intrinsic binding enthalpy of lipid to DNA as +2.8+/-0.3 kJ/mol. The pK(a) of DOPE was calculated to shift from 7.7+/-0.1 in the free state to 8.8+/-0.1 in the complex. At physiological ionic strength, proton linkage was not observed upon complex formation and the buffer-independent binding enthalpy was +1.0+/-0.4 kJ/mol. These studies indicate that the intrinsic interaction between 1:1 DOTAP/DOPE and DNA is an entropy-driven process and that the affinities of cationic lipids that are formulated with and without DOPE for DNA are controlled by the positive entropic changes that occur upon complex formation.     

Calorimetric determination of linkage effects involving an acyl-enzyme intermediate

Enthalpy changes of alpha-chymotrypsin acylation by 3-(2-furyl)acryloylimidazole (FAI) were calorimetrically determined as a function of pH. By observing the functional dependence of acylation enthalpies on buffer ionization heats, a complex pH profile was obtained describing proton release accompanying formation of acyl-enzyme. A pKa of 4.0 for FAI ionization and apparent pKa values of 6.8, 7.55 and 8.8 on the enzyme were used to account for the proton release data. A model which accounts for the proton release behavior was used to fit the acylation enthalpy data and values for the apparent dissociation enthalpies of the groups involved were obtained along with a pH-independent intrinsic enthalpy of acylation. This model suggests a group with an apparent pK = 6.8 and delta Hion = 8.7 kcal/mol which is perturbed to a pK of 7.55 and delta Hion = 7.6 kcal/mol on attachment of the acyl moiety to the enzyme. The apparent ionization enthalpy change for the active-inactive transition (pK3 = 8.8; delta H = 3.0 kcal/mol) corresponds with that calculated from the data of Fersht (J. Mol. Biol. 64 (1972) 497). The pH-independent intrinsic enthalpy of acylation (delta H = -7.9 kcal/mol) is corrected for group ionizations linked to the acylation process. Consequently, it more closely reflects molecular processes of interest such as substrate binding, covalent bond rearrangement, and product release.     

Sequence-specific DNA binding by the glucocorticoid receptor DNA-binding domain is linked to a salt-dependent histidine protonation

We used isothermal titration calorimetry in the temperature range 21-25 degrees C to investigate the effect of pH on the calorimetric enthalpy (delta H(cal)) for sequence specific DNA-binding of the glucocorticoid receptor DNA-binding domain (GR DBD). Titrations were carried out in solutions containing 100 mM NaCl, 1 mM dithiothreitol, 5% glycerol by volume, and 20 mM Tris, Hepes, Mops, or sodium phosphate buffers at pH 7.5. A strong dependence of delta H(cal) on the buffer ionization enthalpy is observed, demonstrating that the DNA binding of the GR DBD is linked to proton uptake at these conditions. The apparent increase in the pK(a) for an amino acid side chain upon DNA binding is supported by the results of complementary titrations, where delta H(cal) shows a characteristic dependence on the solution pH. delta H(cal) is also a function of the NaCl concentration, with opposite dependencies in Tris and Hepes buffers, respectively, such that a similar delta H(cal) value is approached at 300 mM NaCl. This behavior shows that the DNA-binding induced protonation is inhibited by increased concentrations of NaCl. A comparison with structural data suggests that the protonation involves a histidine (His451) in the GR DBD, because in the complex this residue is located close to a DNA phosphate at an orientation that is consistent with a charged-charged hydrogen bond in the protonated state. NMR spectra show that His451 is not protonated in the unbound protein at pH 7.5. The pH dependence in delta H(cal) can be quantitatively described by a shift of the pK(a) of His451 from approximately 6 in the unbound state to close to 8 when bound to DNA at low salt concentration conditions. A simple model involving a binding competition between a proton and a Na(+) counterion to the GR DBD-DNA complex reproduces the qualitative features of the salt dependence.     

Energetics of Ca(2+)-EDTA interactions: calorimetric study

The interaction between Ca(2+) and EDTA has been studied using isothermal titration calorimetry to elucidate the detailed mechanism of complex formation and to relate the apparent thermodynamic parameters of calcium binding to the intrinsic effects of ionization. It has been shown that Ca(2+) binding to EDTA is an exothermic process in the temperature range 5-48 degrees C and is highly dependent on the buffer in which the reaction occurs. Calorimetric measurements along with pH-titration of EDTA under different solvent conditions shows that the apparent enthalpy effect of the binding is predominantly from the protonation of buffer. Subtraction of the ionization effect of buffer from the total enthalpy shows that the enthalpy of binding Ca(2+) to EDTA is -0.56 kcal mol(-1) at pH 7.5. The DeltaH value strongly depends on solvent conditions as a result of the degree of ionization of the two amino groups in the EDTA molecule, but depends little on temperature, indicating that the heat capacity increment for metal binding is close to zero. At physiological pH values where the amino groups of EDTA with pK(a)=6.16 and pK(a)=10.26 are differently ionized, the coordination of the Ca(2+) ion into the complex leads to release of one proton due to deprotonation of the amino group having pK(a)=10.26. Increasing the pH up to 11.2, where little or no ionization occurs, leads to elimination of the enthalpy component due to ionization, while its decrease to pH 2 leads to its increase, due to protonation of the two amino groups. The heat effect of Ca(2+)/EDTA interactions, excluding the deprotonation enthalpy of the amino groups, i.e. that associated with the intrinsic enthalpy of binding, is higher in value (Delta(b)H(o)=-5.4 kcal mol(-1)) than the apparent enthalpy of binding. Thus, the large DeltaG value for Ca(2+) binding to EDTA arises not only from favorable entropic but also enthalpic changes, depending on the ionization state of the amino groups involved in coordination of the calcium. This explains the great variability in DeltaH obtained in previous studies. The ionization enthalpy is always unfavorable, and therefore dramatically decreases Ca(2+) affinity by reduction of the enthalpy term of the stability function. The origin of the enthalpy and entropy terms in the stability of the Ca(2+)-EDTA complex is discussed.     

Structural parameterization of the binding enthalpy of small ligands

A major goal in ligand and drug design is the optimization of the binding affinity of selected lead molecules. However, the binding affinity is defined by the free energy of binding, which, in turn, is determined by the enthalpy and entropy changes. Because the binding enthalpy is the term that predominantly reflects the strength of the interactions of the ligand with its target relative to those with the solvent, it is desirable to develop ways of predicting enthalpy changes from structural considerations. The application of structure/enthalpy correlations derived from protein stability data has yielded inconsistent results when applied to small ligands of pharmaceutical interest (MW < 800). Here we present a first attempt at an empirical parameterization of the binding enthalpy for small ligands in terms of structural information. We find that at least three terms need to be considered: (1) the intrinsic enthalpy change that reflects the nature of the interactions between ligand, target, and solvent; (2) the enthalpy associated with any possible conformational change in the protein or ligand upon binding; and, (3) the enthalpy associated with protonation/deprotonation events, if present. As in the case of protein stability, the intrinsic binding enthalpy scales with changes in solvent accessible surface areas. However, an accurate estimation of the intrinsic binding enthalpy requires explicit consideration of long-lived water molecules at the binding interface. The best statistical structure/enthalpy correlation is obtained when buried water molecules within 5-7 A of the ligand are included in the calculations. For all seven protein systems considered (HIV-1 protease, dihydrodipicolinate reductase, Rnase T1, streptavidin, pp60c-Src SH2 domain, Hsp90 molecular chaperone, and bovine beta-trypsin) the binding enthalpy of 25 small molecular weight peptide and nonpeptide ligands can be accounted for with a standard error of +/-0.3 kcal x mol(-1).     

Proton exchange coupled to the specific binding of alkylsulfonates to serum albumins

We have applied isothermal titration calorimetry to investigate the linkage between ligand binding and the uptake or release of protons by human serum albumin (HSA) and bovine serum albumin (BSA). The ligands were sodium decyl sulfate (SDeS) and sodium dodecyl sulfate (SDS). Within a certain temperature range, the binding isotherm could be clearly resolved into two classes of sites (high affinity and low affinity) and modeled assuming independence and thermodynamic equivalence of the sites within each class. Measurements at pH 7.0 in different buffer systems revealed that the binding of SDS to the high affinity sites did not couple to any exchange of protons in either of the proteins. Saturation of the 6-8 low affinity sites for SDS, on the other hand, brought about the release of two protons from both HSA and BSA. In addition to elucidating the pH dependence of ligand binding, this analysis stressed that binding enthalpies for the low affinity sites measured by calorimetry must be corrected for effects due to the concomitant protonation of the buffer. The shorter ligand SDeS bound to HSA with a comparable stoichiometry but with four times lower affinity. Interestingly, no proton linkage was observed for the binding of SDeS. An empirical structural analysis suggested that His 242 in site 7 (of HSA) is a likely candidate for one of the proton donors.     

Binding Thermodynamics of Ferredoxin:NADP Reductase: Two Different Protein Substrates and One Energetics

The thermodynamics of the formation of binary and ternary complexes between Anabaena PCC 7119 FNR and its substrates, NADP⁺ and Fd, or Fld, has been studied by ITC. Despite structural dissimilarities, the main difference between Fd and Fld binding to FNR relates to hydrophobicity, reflected in different binding heat capacity and number of water molecules released from the interface. At pH 8, the formation of the binary complexes is both enthalpically and entropically driven, accompanied by the protonation of at least one ionizable group. His²⁹⁹ FNR has been identified as the main responsible for the proton exchange observed. However, at pH 10, where no protonation occurs and intrinsic binding parameters can be obtained, the formation of the binary complexes is entropically driven, with negligible enthalpic contribution. Absence of the FMN cofactor in Fld does not alter significantly the strength of the interaction, but considerably modifies the enthalpic and entropic contributions, suggesting a different binding mode. Ternary complexes show negative cooperativity (6-fold and 11-fold reduction in binding affinity, respectively), and an increase in the enthalpic contribution (more favorable) and a decrease in the entropic contribution (less favorable), with regard to the binary complexes energetics.     

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

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

Microcalorimetric study of wheat germ agglutinin binding to N-acetylglucosamine and its oligomers.

The energetics of association of wheat germ agglutinin (WGA) with N-acetylglucosamine (GlcNAc) and its beta(1,4) oligomers have been measured using isothermal titration calorimetry. Association constants of 0.4, 5.3, 11.1, 12.3, and 19.1 mM-1 and enthalpies of binding of -6.1, -15.6, -19.4, -19.3, and -18.2 kcal mol-1 were obtained at 26 degrees C for the titration of WGA with GlcNAc, (GlcNAc)2, (GlcNAc)3, (GlcNAc)4, and (GlcNAc)5, respectively. The term T delta S was always of negative value, indicating that the binding process is enthalpically driven. Titrations of WGA performed at pH 4.5 did not differ significantly from those performed at pH 7.0, suggesting that no groups with a pKa in this range are directly involved in the binding event. Also, performing the titration in a buffer system with a higher enthalpy of protonation did not change the enthalpy of binding confirming that there is no net protonation or deprotonation when WGA binds GlcNAc residues at pH 7. A model of four independent binding sites was found to adequately describe the binding curves, except in the case of (GlcNAc)4 which exhibited positive cooperativity. The energetic values are discussed within the context of the structure of the WGA-(GlcNAc)2 complex.     

Energetics of primary processes in visula escitation: photocalorimetry of rhodopsin in rod outer segment membranes.

A sensitive technique for the direct calorimetric determination of the energetics of photochemical reactions under low levels of illumination, and its application to the study of primary processes in visula excitation, are described. Enthlpies are reported for various steps in the bleaching of rhodopsin in intact rod outer segment membranes, together with the heats of appropriate model reactions. Protonation changes are also determined calorimetrically by use of buffers with differing heats of proton ionization. Bleaching of rhodopsin is accompanied by significant uptake of heat energy, vastly in excess of the energy required for simple isomerization of the retinal chromophore. Metarhodopsin I formation involves the uptake of about 17 kcal/mol and no net change in proton ionization of the system. Formation of metarhodopsin II requires an additional energy of about 10 kcal/mol and involves the uptake on one hydrogen ion from solution. The energetics of the overall photolysis reaction, rhodopsin leads to opsin + all-trans-retinal, are pH dependent and involve the exposure of an additional titrating group on opsin. This group has a heat of proton ionization of about 12 kcal/mal, characteristic of a primary amine, but a pKa in the region of neutrality. We suggest that this group is the Schiff base lysine of the chromophore binding site of rhodopsin which becomes exposed on photolysis. The low pKa for this active lysine would result in a more stable retinal-opsin linkage, and might be induced by a nearby positively charged group on the protein (either arginine or a second lysine residue). This leads to a model involving intramolecular protonation of the Schiff base nitrogen in the retinal-opsin linkage of rhodopsin, which is consistent with the thermodynamic and spectroscopic properties of the system. We further propose that the metarhodopsin I leads to metarhodopsin II step in the bleaching sequence involves reversible hydrolysis of the Schiff base linkage in the chromophore binding site, and that subsequent steps are the result of migration of the chromophore from this site.     

On the link between conformational changes,ligand binding and heat capacity

BackgroundConformational changes coupled to ligand binding constitute the structural and energetics basis underlying cooperativity, allostery and, in general, protein regulation. These conformational rearrangements are associated with heat capacity changes. ITC is a unique technique for studying binding interactions because of the simultaneous determination of the binding affinity and enthalpy, and for providing the best estimates of binding heat capacity changes.Scope of reviewStill controversial issues in ligand binding are the discrimination between the “conformational selection model” and the “induced fit model”, and whether or not conformational changes lead to temperature dependent apparent binding heat capacities. The assessment of conformational changes associated with ligand binding by ITC is discussed. In addition, the “conformational selection” and “induced fit” models are reconciled, and discussed within the context of intrinsically (partially) unstructured proteins.Major conclusionsConformational equilibrium is a major contribution to binding heat capacity changes. A simple model may explain both conformational selection and induced fit scenarios. A temperature-independent binding heat capacity does not necessarily indicate absence of conformational changes upon ligand binding. ITC provides information on the energetics of conformational changes associated with ligand binding (and other possible additional coupled equilibria).General significancePreferential ligand binding to certain protein states leads to an equilibrium shift that is reflected in the coupling between ligand binding and additional equilibria. This represents the structural/energetic basis of the widespread dependence of ligand binding parameters on temperature, as well as pH, ionic strength and the concentration of other chemical species. This article is part of a Special Issue entitled Microcalorimetry in the BioSciences — Principles and Applications, edited by Fadi Bou-Abdallah.     

Protonation changes upon ligand binding to trypsin and thrombin: structural interpretation based on pK(a) calculations and ITC experiments

The protonation states of a protein and a ligand can be altered upon complex formation. Such changes can be detected experimentally by isothermal titration calorimetry (ITC). For a series of ligands binding to the serine proteases trypsin and thrombin, we previously performed an extensive ITC and crystallographic study and were able to identify protonation changes for four complexes. However, since ITC measures only the overall proton exchange, it does not provide structural insights into the functional groups involved in the proton transfer. Using Poisson-Boltzmann calculations based on our recently developed PEOE_PB charges, we compute pK(a) values for all complexes of our former study in order to reveal the residues with altered protonation states. The results indicate that His57, a member of the catalytic triad, is responsible for the most relevant pK(a) shifts leading to the experimentally detected protonation changes. This finding is in contrast to our previous assumption that the observed protonation changes occur at the carboxylic group of the ligands. The newly detected proton acceptor is used for a revised factorization of the ITC data, which is necessary whenever the protonation inventory changes upon complexation. The pK(a) values of complexes showing no protonation change in the ITC experiment are reliably predicted in most cases, whereas predictions of strongly coupled systems remain problematic.     

Structural energetics of protein folding and binding

Structural energetics is a method for calculating the energetics of protein folding and binding reactions as a function of temperature. This approach allows measured energetics to be interpreted with regards to the protein structure and the prediction of energetics from known structures. Recent advances include improvements in the parameterization of enthalpy, entropy and heat capacity terms and new applications, especially with regards to understanding dynamic properties of proteins and how these are affected by ligand binding.     

Energetics of subunit dimerization in bacteriophage lambda cI repressor: linkage to protons, temperature, and KCl.

A common feature of gene regulatory systems is the linkage between reversible protein oligomerization and DNA binding. Experimental dissection using temperature dependence of the subunit-subunit energetics and their linkage to processes such as ion binding and release is necessary for characterization of the chemical forces that contribute to cooperativity and site specificity. We have therefore studied the effects of temperature, proton activity, and monovalent salt on monomer-dimer assembly of the lambda cI repressor using a recently developed gel chromatographic procedure. This technique has made possible studies in the previously inaccessible picomolar concentration ranges where the assembly reactions occur. Upon formation of the dimer interface in the range pH 5-9, we find an overall absorption of protons which is temperature-dependent. The dimerization reaction displays a large negative enthalpy of association at all conditions studied (pH 5, 7, and 9). The reaction is also dependent on monovalent salt concentration: subunit association is weaker at low-salt conditions. The results suggest that a repulsive interaction between negatively charged side chains (i.e., aspartates and glutamates) on each monomer surface is attenuated by increasing concentrations of KCl. Formation of the dimer interface may be mediated by absorption of cations which stabilize the complex.