Interaction kinetics of tetramethylrhodamine transferrin with human transferrin receptor studied by fluorescence correlation spectroscopy.

We applied fluorescence correlation spectroscopy (FCS) to characterize the interaction dynamics of fluorescence-labeled transferrin with transferrin receptor (hTfR) associates isolated from human placenta. The dissociation constant for the equilibrium binding of TMR-labeled ferri-transferrin to hTfR in detergent free solution was determined to be 7 +/- 3 nM. Binding curves were compatible with equal and independent binding sites present on the hTfR associates. Under pseudo-first-order conditions, with respect to transferrin, complex formation is monophasic. From these curves, association and dissociation rate constants for a reversible bimolecular binding reaction were determined, with (1.1 +/- 0.1) x 10(4) M-1 s-1 for the former and (6 +/- 4) x 10(-)4 s-1 for the latter. In dissociation exchange experiments, biphasic curves and concentration-independent reciprocal relaxation times were determined. From isothermal titration calorimetry experiments, we obtained an enthalpy change of -44.4 kJ/mol associated with the reaction. We thus conclude that the reaction is mainly enthalpy driven.     

Dipole-dipole interaction stabilizes the transition state of apurinic/apyrimidinic endonuclease--abasic site interaction

Human apurinic/apyrimidinic (AP) endonuclease (hAPE) initiates the repair of an abasic site (AP site). To gain insight into the mechanisms of damage recognition of hAPE, we conducted surface plasmon resonance spectroscopy to study the thermodynamics and kinetics of its interaction with substrate DNA containing an abasic site (AP DNA). The affinity of hAPE binding toward DNA increased as much as 6-fold after replacing a single adenine (equilibrium dissociation constant, K(D), 5.3 nm) with an AP site (K(D), 0.87 nm). The enzyme-substrate complex formation appears to be thermodynamically stabilized and favored by a large change in Gibbs free energy, DeltaG degrees (-50 kJ/mol). The latter is supported by a high negative change in enthalpy, DeltaH degrees (-43 kJ/mol) and also positive change in entropy, DeltaS degrees (24 J/(K mol)), and thus the binding process is spontaneous at all temperatures. Analysis of kinetic parameters reveals small enthalpy of activation for association, DeltaH degrees++(ass) (-17 kJ/mol), and activation energy for association (E(a), -14 kJ/mol) when compared with the enthalpy of activation for dissociation, DeltaH degrees++(diss) (26 kJ/mol), and activation energy in the reverse direction (E(d), 28 kJ/mol). Furthermore, varying concentration of KCl showed an increase in binding affinity at low concentration but complete abrogation of the binding at higher concentration, implying the importance of hydrophobic, but predominantly ionic, forces in the Michaelis-Menten complex formation. Thus, low activation energy and the enthalpy of activation, which are perhaps a result of dipole-dipole interactions, play critical roles in AP site binding of APE.     

Thermodynamics of the arginine kinase reaction.

The effect of temperature, pH, free [Mg(2+)], and ionic strength on the apparent equilibrium constant of arginine kinase (EC 2.7.3.3) was determined. At equilibrium, the apparent K' was defined as [see text] where each reactant represents the sum of all the ionic and metal complex species. The K' at pH 7.0, 1.0 mM free [Mg(2+)], and 0. 25 M ionic strength was 29.91 +/- 0.59, 33.44 +/- 0.46, 35.44 +/- 0. 71, 39.64 +/- 0.74, and 45.19 +/- 0.65 (n = 8) at 40, 33, 25, 15, and 5 degrees C, respectively. The standard apparent enthalpy (DeltaH degrees') is -8.19 kJ mol(-1), and the corresponding standard apparent entropy of the reaction (DeltaS degrees') is + 2. 2 J K(-1)mol(-1) in the direction of ATP formation at pH 7.0, free [Mg(2+)] =1.0 mM, ionic strength (I) =0.25 M at 25 degrees C. We further show that the magnitude of transformed Gibbs energy (DeltaG degrees ') of -8.89 kJ mol(-1) is mostly comprised of the enthalpy of the reaction, with 7.4% coming from the entropy TDeltaS degrees' term (+0.66 kJ mol(-1)). Our results are discussed in relation to the thermodynamic properties of its evolutionary successor, creatine kinase.     

Thermodynamic parameters and shape of the mycobacterial polymethylpolysaccharide-fatty acid complex

Properties of the mycobacterial polymethylpolysaccharide-lipid complex have been investigated by fluorometric techniques. From the dissociation constant for the O-methyglucose polysaccharide-parinaric acid complex at 293 K, a Gibbs free energy (delta G degree) of -33.65 kJ/mol was obtained. The Kd decreased with increasing temperature, giving an enthalpy (delta H degree) of 15.4 kJ/mol. From these data, a molar entropy (delta S degree) of 167.4 J K-1 was obtained. Thus, the reaction is slightly endothermic, but the large positive entropy change leads to an overall negative free energy favoring complex formation. From fluorescence depolarization measurements, the methylglucose polysaccharide-parinaric acid complex appears to display isotropic rotation with a correlation time of 2.55 ns at 23 degrees C. This may be compared to a rotational correlation time of 6.17 ps for free parinaric acid in water at 23 degrees C calculated from the value determined in cyclohexanol at the same temperature, which demonstrates that the mobility of the fatty acid in the complex is restricted. Assuming the complex is spherical, it was calculated to have a diameter of 23-26 A, whereas a helical methyglucose polysaccharide molecule assembled from space-filling models has the dimensions of a cylinder of 18 X 24 A. The polysaccharide and fatty acid chain-length dependence of the interaction shows a discontinuity for helical polysaccharide segments shorter than 12 sugars and for fatty acids shorter than palmitate.     

Temperature dependence of the nucleation constant rate in beta amyloid fibrillogenesis

Beta-amyloid peptide (A beta), in fibrillar form, is the primary constituent of senile plaques, a defining feature of Alzheimer's disease (AD). In solution assays, fibrils form with a lag time, interpreted as a nucleation/condensation-dependent process. The kinetics of fibrillogenesis is controlled by two key parameters: nucleation and elongation rate constants. We report here the study of the temperature dependence of the nucleation rate constant on an A beta monomer concentration of 18.4 microM at pH 7.4 and at temperatures ranging from 302 to 318 K. We found that the nucleation constant varied as in the Arrhenius law, giving an activation energy of 311.2 kJ mol(-1). The corresponding values of enthalpy of activation (deltaH*), entropy of activation (deltaS*) and Gibbs energy of activation (deltaG*) were evaluated by Eyring's equation of absolute reaction rate. A Gibbs energy of activation of approximately 110 kJ mol(-1) was obtained.     

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)     

Energetics of syntrophic propionate oxidation in defined batch and chemostat cocultures

Propionate consumption was studied in syntrophic batch and chemostat cocultures of Syntrophobacter fumaroxidans and Methanospirillum hungatei. The Gibbs free energy available for the H(2)-consuming methanogens was <-20 kJ mol of CH(4)(-1) and thus allowed the synthesis of 1/3 mol of ATP per reaction. The Gibbs free energy available for the propionate oxidizer, on the other hand, was usually >-10 kJ mol of propionate(-1). Nevertheless, the syntrophic coculture grew in the chemostat at steady-state rates of 0.04 to 0. 07 day(-1) and produced maximum biomass yields of 2.6 g mol of propionate(-1) and 7.6 g mol of CH(4)(-1) for S. fumaroxidans and M. hungatei, respectively. The energy efficiency for syntrophic growth of S. fumaroxidans, i.e., the biomass produced per unit of available Gibbs free energy was comparable to a theoretical growth yield of 5 to 12 g mol of ATP(-1). However, a lower growth efficiency was observed when sulfate served as an additional electron acceptor, suggesting inefficient energy conservation in the presence of sulfate. The maintenance Gibbs free energy determined from the maintenance coefficient of syntrophically grown S. fumaroxidans was surprisingly low (0.14 kJ h(-1) mol of biomass C(-1)) compared to the theoretical value. On the other hand, the Gibbs free-energy dissipation per mole of biomass C produced was much higher than expected. We conclude that the small Gibbs free energy available in many methanogenic environments is sufficient for syntrophic propionate oxidizers to survive on a Gibbs free energy that is much lower than that theoretically predicted.     

Energetics of heparin binding to human acidic fibroblast growth factor

The binding of low-molecular-weight heparin to an amino-terminal-truncated, 132-amino-acid, human acidic fibroblast growth factor form has been studied by isothermal titration calorimetry. This technique yields values for the enthalpy change and equilibrium constant, from which the Gibbs energy and entropy change are also calculated. Experiments in different buffers and pH values show that the protonic balance during the reaction is negligible. Experiments made at pH 7.0 with NaCl concentrations ranging from 0.20 to 0.60 M revealed changes in enthalpy and Gibbs energy in the range of -30- -17 and -27- -24 kJ x mol(-1), respectively. Isothermal titration calorimetry was also performed at different temperatures to obtain a value for the heat-capacity change at pH 7.0 and 0.4 M NaCl concentration of -96 J K- x mol(-1). A change in the length of heparin brought about no change in the thermodynamic parameters at 25 degrees C under the same experimental conditions. Changes upon ligand binding in the range of -50- -200 A2 in both polar and non-polar solvent-accessible surface areas were calculated from thermodynamic data by using different parametric equations taken from the literature. These values suggest a negligible overall conformational change in the protein when it binds to heparin and no formation of any protein-protein interface.     

A calorimetric investigation of the interaction of the lac repressor with inducer

A calorimetric study has been made of the interaction between the lac repressor and isopropyl-1-thio-beta-D-galactopyranoside (IPTG). The buffer-corrected enthalpy of reaction at 25 degrees C was found to be -15.6, -24.7, -4.6 kJ/mol of bound IPTG at pH 7.0, pH 8.1, and pH 9.0, respectively. This large range of enthalpy values is in contrast to a maximum difference in the free energy of the reaction of only 1.5 kJ/mol of bound IPTG between these pH values. The reaction was found by calorimetric measurements in different buffers to be accompanied by an uptake of 0.29 mol of protons/mol of bound IPTG at pH 8.1. The pH dependency of the reaction enthalpy suggests differences in the extent of protonation of the binding site and the involvement of H bonding with IPTG. The lack of strong hydrophobic contributions in the IPTG binding process is revealed by the absence of any determinable heat capacity change for the reaction at pH 7.0. The presence of phosphate buffer significantly alters the enthalpy of IPTG binding at higher pH values, but has little effect upon the binding constant. This implies that highly negative phosphate species change the nature of the IPTG binding site without any displacement of phosphate upon IPTG binding.     

The energetics of HMG box interactions with DNA: thermodynamics of the DNA binding of the HMG box from mouse sox-5

The energetics of the Sox-5 HMG box interaction with DNA duplexes, containing the recognition sequence AACAAT, were studied by fluorescence spectroscopy, isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC). Fluorescence titration showed that the association constant of this HMG box with the duplexes is of the order 4x10(7) M(-1), increasing somewhat with temperature rise, i.e. the Gibbs energy is -40 kJ mol(-1) at 5 degrees C, decreasing to -48 kJ mol(-1) at 32 degrees C. ITC measurements of the enthalpy of association over this temperature range showed an endothermic effect below 17 degrees C and an exothermic effect above, suggesting a heat capacity change on binding of about -4 kJ K(-1) mol(-1), a value twice larger than expected from structural considerations. A straightforward interpretation of ITC data in heat capacity terms assumes, however, that the heat capacities of all participants in the association reaction do not change over the considered temperature range. Our previous studies showed that over the temperature range of the ITC experiments the HMG box of Sox-5 starts to unfold, absorbing heat and the heat capacities of the DNA duplexes also increase significantly. These heat capacity effects differ from that of the DNA/Sox-5 complex. Correcting the ITC measured binding enthalpies for the heat capacity changes of the components and complex yielded the net enthalpies which exhibit a temperature dependence of about -2 kJ K(-1) mol(-1), in good agreement with that predicted on the basis of dehydration of the protein-DNA interface. Using the derived heat capacity change and the enthalpy and Gibbs energy of association measured at 5 degrees C, the net enthalpy and entropy of association of the fully folded HMG box with the target DNA duplexes was determined over a broad temperature range. These functions were compared with those for other known cases of sequence specific DNA/protein association. It appears that the enthalpy and entropy of association of minor groove binding proteins are more positive than for proteins binding in the major groove. The observed thermodynamic characteristics of protein binding to the A+T-rich minor groove of DNA might result from dehydration of both polar and non-polar groups at the interface and release of counterions. The expected entropy of dehydration was calculated and found to be too large to be compensated by the negative entropy of reduction of translational/rotational freedom. This implies that DNA/HMG box association proceeds with significant decrease of conformational entropy, i.e. reduction in conformational mobility.     

Stability studies on derivatives of the bovine pancreatic trypsin inhibitor

Gibbs energy, enthalpy, and entropy data were determined for two selectively modified analogues of bovine pancreatic trypsin inhibitor (BPTI) to provide a model free set of thermodynamic parameters that characterize (a) the energetic and entropic contributions of the 14-38 disulfide bridge and (b) the variation of the overall stability resulting from the introduction of two negative charges into the positions 14 and 38. The two BPTI analogues studied were BPTI having Cys-14 and Cys-38 carboxymethylated (BPTI-RCOM) and BPTI having Cys-14 and Cys-38 carboxamidomethylated (BPTI-RCAM). They were obtained from native BPTI by reduction, followed by modification of the sulfhydryl groups with iodoacetic acid or iodoacetamide, respectively. The temperature dependence of all thermodynamic parameters of BPTI is drastically altered in the absence of the third disulfide bridge. Even the apparently minute difference of two dissociable carboxyl groups instead of uncharged amide groups in positions 14 and 38 has surprisingly large effects on the temperature dependence of the stabilization enthalpy. The Gibbs energy of BPTI at pH 2, 25 degrees C, decreases by approximately 70% when the 14-38 disulfide bond is cleaved. BPTI-RCOM is more stable than BPTI-RCAM in the whole pH range studied. The difference of -4 kJ/mol at pH 2, 25 degrees C, is reduced to -2.7 kJ/mol at pH 5, 25 degrees C. This finding demonstrates that the presence of two negative charges reduces the higher stability of BPTI-RCOM slightly; however, the overall effect of the two charges is still a stabilization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stability and DNA binding ability of the DNA binding domains of interferon regulatory factors 1 and 3

The thermodynamic properties and DNA binding ability of the N-terminal DNA binding domains of interferon regulatory factors IRF-1 (DBD1) and IRF-3 (DBD3) were studied using microcalorimetric and optical methods. DBD3 is significantly more stable than DBD1: at 20 degrees C the Gibbs energy of unfolding of DBD3 is -28.6 kJ/mol, which is 2 times larger than that of DBD1, -14.9 kJ/mol. Fluorescence anisotropy titration experiments showed that at this temperature the association constants with the PRDI binding site are 1.1 x 10(6) M(-)(1) for DBD1 and 3.6 x 10(6) M(-)(1) for DBD3, corresponding to Gibbs energies of association of -34 and -37 kJ/mol, respectively. However, the larger binding energy of DBD3 is due to its larger electrostatic component, while its nonelectrostatic component is smaller than that of DBD1. Therefore, DBD1 appears to have more sequence specificity than DBD3. Binding of DBD1 to target DNA is characterized by a substantially larger negative enthalpy than binding of DBD3, implying that the more flexible structure of DBD1 forms tighter contacts with DNA than the more rigid structure of DBD3. Thus, the strength of the DBDs' specific association with DNA is inversely related to the stability of the free DBDs.     

Energetics of myo-inositol hexasulfate binding to human acidic fibroblast growth factor effect of ionic strength and temperature.

The binding of myo-inositol hexasulfate to an N-terminal truncated 132-amino-acid human acidic fibroblast growth factor form was studied by isothermal titration calorimetry. The technique yields values for the enthalpy change and equilibrium constant, from which the Gibbs energy and entropy change can also be calculated. Experiments in different buffers and pH values show that the proton balance in the reaction is negligible. Experiments at pH 7.0 in the presence of 0.2-0.6 M NaCl showed that the enthalpy and Gibbs energy changes parallel behaviour with ionic strength change, with values in the -21 to -11 kJ x mol(-1) range in the first case and in the -31 to -22 kJ x mol(-1) range in the second. No dependence of entropy on ionic strength was found, with a constant value of approximately 35 J x K(-1) x mol(-1) at all ionic strengths studied. The results can be interpreted in molecular terms by a model in which competitive binding of 3-4 chloride ions to the myo-inositol-binding site is assumed. Isothermal titration calorimetry was also performed at different temperatures and yielded a value of -142+/-13 J x K(-1) x mol(-1) for the heat-capacity change at pH 7.0 and 0.4 M NaCl. Using different parametric equations in the literature, changes on ligand binding in the range -100 to -200 A2 in solvent-accessible surface areas, both polar and apolar, were calculated from thermodynamic data. These values suggest a negligible overall conformational change in the protein when the ligand binds and agree closely with calculations performed with NMR structural data, in which it is shown that the most important negative change in total solvent-accessible surface area occurs in the amino acids Ile56, Gln57, Leu58 and Leu149, in the high-affinity receptor-binding region of the protein.     

Reductive unfolding of serum albumins uncovered by Raman spectroscopy

The reductive unfolding of bovine serum albumin (BSA) and human serum albumin (HSA) induced by dithiothreitol (DTT) is investigated using Raman spectroscopy. The resolution of the S-S Raman band into both protein and oxidized DTT contributions provides a reliable basis for directly monitoring the S-S bridge exchange reaction. The related changes in the protein secondary structure are identified by analyzing the protein amide I Raman band. For the reduction of one S-S bridge of BSA, a mean Gibbs free energy of -7 kJ mol(-1) is derived by studying the reaction equilibrium. The corresponding value for the HSA S-S bridge reduction is -2 kJ mol(-1). The reaction kinetics observed via the S-S or amide I Raman bands are identical giving a reaction rate constant of (1.02 +/- 0.11) M(-1) s(-1) for BSA. The contribution of the conformational Gibbs free energy to the overall Gibbs free energy of reaction is further estimated by combining experimental data with ab initio calculations.     

Catalytic mechanism of SGAP, a double-zinc aminopeptidase from Streptomyces griseus

The catalytic mechanism underlying the aminopeptidase from Streptomyces griseus (SGAP) was investigated. pH-dependent activity profiles revealed the enthalpy of ionization for the hydrolysis of leucine-para-nitroanilide by SGAP. The value obtained (30 +/- 5 kJ.mol(-1)) is typical of a zinc-bound water molecule, suggesting that the zinc-bound water/hydroxide molecule acts as the reaction nucleophile. Fluoride was found to act as a pure noncompetitive inhibitor of SGAP at pH values of 5.9-8 with a K(i) of 11.4 mM at pH 8.0, indicating that the fluoride ion interacts equally with the free enzyme as with the enzyme-substrate complex. pH-dependent pK(i) experiments resulted in a pK(a) value of 7.0, suggesting a single deprotonation step of the catalytic water molecule to an hydroxide ion. The number of proton transfers during the catalytic pathway was determined by monitoring the solvent isotope effect on SGAP and its general acid-base mutant SGAP(E131D) at different pHs. The results indicate that a single proton transfer is involved in catalysis at pH 8.0, whereas two proton transfers are implicated at pH 6.5. The role of Glu131 in binding and catalysis was assessed by determining the catalytic constants (K(m), k(cat)) over a temperature range of 293-329 degrees K for both SGAP and the E131D mutant. For the binding step, the measured and calculated thermodynamic parameters for the reaction (free energy, enthalpy and entropy) for both SGAP and the E131D mutant were similar. By contrast, the E131D point mutation resulted in a four orders of magnitude decrease in k(cat), corresponding to an increase of 9 kJ.mol(-1) in the activation energy for the E131D mutant, emphasizing the crucial role of Glu131 in catalysis.     

Application of the thermorheologically complex nonlinear Adam-Gibbs model for the glass transition to molecular motion in hydrated proteins

The nonlinear thermorheologically complex Adam Gibbs (extended "Scherer-Hodge") model for the glass transition is applied to enthalpy relaxation data reported by Sartor, Mayer, and Johari for hydrated methemoglobin. A sensible range in values for the average localized activation energy is obtained (100-200 kJ mol(-1)). The standard deviation in the inferred Gaussian distribution of activation energies, computed from the reported KWW beta-parameter, is approximately 30% of the average, consistent with the suggestion that some relaxation processes in hydrated proteins have exceptionally low activation energies.     

Thermodynamic dissection of the binding energetics of KNI-272, a potent HIV-1 protease inhibitor

KNI-272 is a powerful HIV-1 protease inhibitor with a reported inhibition constant in the picomolar range. In this paper, a complete experimental dissection of the thermodynamic forces that define the binding affinity of this inhibitor to the wild-type and drug-resistant mutant V82F/184V is presented. Unlike other protease inhibitors, KNI-272 binds to the protease with a favorable binding enthalpy. The origin of the favorable binding enthalpy has been traced to the coupling of the binding reaction to the burial of six water molecules. These bound water molecules, previously identified by NMR studies, optimize the atomic packing at the inhibitor/protein interface enhancing van der Waals and other favorable interactions. These interactions offset the unfavorable enthalpy usually associated with the binding of hydrophobic molecules. The association constant to the drug resistant mutant is 100-500 times weaker. The decrease in binding affinity corresponds to an increase in the Gibbs energy of binding of 3-3.5 kcal/mol, which originates from less favorable enthalpy (1.7 kcal/mol more positive) and entropy changes. Calorimetric binding experiments performed as a function of pH and utilizing buffers with different ionization enthalpies have permitted the dissection of proton linkage effects. According to these experiments, the binding of the inhibitor is linked to the protonation/deprotonation of two groups. In the uncomplexed form these groups have pKs of 6.0 and 4.8, and become 6.6 and 2.9 in the complex. These groups have been identified as one of the aspartates in the catalytic aspartyl dyad in the protease and the isoquinoline nitrogen in the inhibitor molecule. The binding affinity is maximal between pH 5 and pH 6. At those pH values the affinity is close to 6 x 10(10) M(-1) (Kd = 16 pM). Global analysis of the data yield a buffer- and pH-independent binding enthalpy of -6.3 kcal/mol. Under conditions in which the exchange of protons is zero, the Gibbs energy of binding is -14.7 kcal/mol from which a binding entropy of 28 cal/K mol is obtained. Thus, the binding of KNI-272 is both enthalpically and entropically favorable. The structure-based thermodynamic analysis indicates that the allophenylnorstatine nucleus of KNI-272 provides an important scaffold for the design of inhibitors that are less susceptible to resistant mutations.     

Thermodynamics and stoicheiometry of the binding of substrate analogues to uricase.

The subunit composition, metal content, substrate-analogue binding and thermal stability of Aspergillus flavus uricase were determined. A. flavus uricase is a tetramer and contains no copper, iron or any other common prosthetic group. Analytical-gel-filtration and equilibrium-dialysis experiments showed one binding site per subunit for urate analogues. The free energy of xanthine binding was -30.5 kJ (-7.3 kcal)/mol of subunit by equilibrium dialysis and -30.1 kJ (-7.2 kcal)/mol of subunit by microcalorimetry. The enthalpy change for xanthine binding was -15.9 kJ (-3.8 kcal)/mol of subunit when determined from the temperature-dependence of the equilibrium constant and -18.0 kJ (-4.3 kcal)/mol of subunit when measured microcalorimetrically. The thermal inactivation rate of A. flavus uricase increases as protein concentration is decreased. This concentration-dependent instability is not due to subunit dissociation.