A calorimetric study on the binding of six general anesthetics to the hydrophobic core of a model protein

The thermodynamic parameters underlying the binding of six volatile general anesthetics to the hydrophobic core of the four-alpha-helix bundle (Aalpha(2)-L38M)(2) are determined using isothermal titration calorimetry. Chloroform, bromoform, trichloroethylene, benzene, desflurane and fluroxene are shown to bind to the four-alpha-helix bundle with dissociation constants of 880+/-10, 90+/-5, 200+/-10, 900+/-30, 220+/-10 and 790+/-40 microM, respectively. The measured dissociation constants for the binding of the six general anesthetics to the four-alpha-helix bundle (Aalpha(2)-L38M)(2) correlate with their human or animal EC(50) values. The negative enthalpy changes indicate that favorable polar interactions are achieved between bound anesthetic and the adjacent amino acid side chains. Because of its small size and the ability to bind a variety of general anesthetics, the four-alpha-helix bundle (Aalpha(2)-L38M)(2) represents an attractive system for structural studies on anesthetic-protein complexes.     

Expression and characterization of a four-alpha-helix bundle protein that binds the volatile general anesthetic halothane

The structural features of volatile anesthetic binding sites on proteins are being investigated with the use of a defined model system consisting of a four-alpha-helix bundle scaffold with a hydrophobic core. The current study describes the bacterial expression, purification, and initial characterization of the four-alpha-helix bundle (Aalpha(2)-L1M/L38M)(2). The alpha-helical content and stability of the expressed protein are comparable to that of the chemically synthesized four-alpha-helix bundle (Aalpha(2)-L38M)(2) reported earlier. The affinity for binding halothane is somewhat improved with a K(d) = 120 +/- 20 microM as determined by W15 fluorescence quenching, attributed to the L1M substitution. Near-UV circular dichroism spectroscopy demonstrated that halothane binding changes the orientation of the aromatic residues in the four-alpha-helix bundle. Nuclear magnetic resonance experiments reveal that halothane binding results in narrowing of the peaks in the amide region of the one-dimensional proton spectrum, indicating that bound anesthetic limits protein dynamics. This expressed protein should prove to be amenable to nuclear magnetic resonance structural studies on the anesthetic complexes, because of its relatively small size (124 residues) and the high affinities for binding volatile anesthetics. Such studies will provide much needed insight into how volatile anesthetics interact with biological macromolecules and will provide guidelines regarding the general architecture of binding sites on central nervous system proteins.     

Binding of volatile anesthetics to serum albumin: measurements of enthalpy and solvent contributions

This study directly examines the enthalpic contributions to binding in aqueous solution of closely related anesthetic haloethers (desflurane, isoflurane, enflurane, and sevoflurane), a haloalkane (halothane), and an intravenous anesthetic (propofol) to bovine and human serum albumin (BSA and HSA) using isothermal titration calorimetry. Binding to serum albumin is exothermic, yielding enthalpies (DeltaH(obs)) of -3 to -6 kcal/mol for BSA with a rank order of apparent equilibrium association constants (K(a) values): desflurane > isoflurane approximately enflurane > halothane >or= sevoflurane, with the differences being largely ascribed to entropic contributions. Competition experiments indicate that volatile anesthetics, at low concentrations, share the same sites in albumin previously identified in crystallographic and photo-cross-linking studies. The magnitude of the observed DeltaH increased linearly with increased reaction temperature, reflecting negative changes in heat capacities (DeltaC(p)). These -DeltaC(p) values significantly exceed those calculated for burial of each anesthetic in a hydrophobic pocket. The enhanced stabilities of the albumin/anesthetic complexes and -DeltaC(p) are consistent with favorable solvent rearrangements that promote binding. This idea is supported by substitution of D(2)O for H(2)O that significantly reduces the favorable binding enthalpy observed for desflurane and isoflurane, with an opposing increase of DeltaS(obs). From these results, we infer that solvent restructuring, resulting from release of water weakly bound to anesthetic and anesthetic-binding sites, is a dominant and favorable contributor to the enthalpy and entropy of binding to proteins.     

Kinetics of anesthetic-induced conformational transitions in a four-alpha-helix bundle protein

Inhaled anesthetics are thought to alter the conformational states of Cys-loop ligand-gated ion channels (LGICs) by binding within discrete cavities that are lined by portions of four alpha-helical transmembrane domains. Because Cys-loop LGICs are complex molecules that are notoriously difficult to express and purify, scaled-down models have been used to better understand the basic molecular mechanisms of anesthetic action. In this study, stopped-flow fluorescence spectroscopy was used to define the kinetics with which inhaled anesthetics interact with (Aalpha(2)-L1M/L38M)(2), a four-alpha-helix bundle protein that was designed to model anesthetic binding sites on Cys-loop LGICs. Stopped-flow fluorescence traces obtained upon mixing (Aalpha(2)-L1M/L38M)(2) with halothane revealed immediate, fast, and slow components of quenching. The immediate component, which occurred within the mixing time of the spectrofluorimeter, was attributed to direct quenching of tryptophan fluorescence upon halothane binding to (Aalpha(2)-L1M/L38M)(2). This was followed by a biexponential fluorescence decay containing fast and slow components, reflecting anesthetic-induced conformational transitions. Fluorescence traces obtained in studies using sevoflurane, isoflurane, and desflurane, which poorly quench tryptophan fluorescence, did not contain the immediate component. However, these anesthetics did produce the fast and slow components, indicating that they also alter the conformation of (Aalpha(2)-L1M/L38M)(2). Cyclopropane, an anesthetic that acts with unusually low potency on Cys-loop LGICs, acted with low apparent potency on (Aalpha(2)-L1M/L38M)(2). These results suggest that four-alpha-helix bundle proteins may be useful models of in vivo sites of action that allow the use of a wide range of techniques to better understand how anesthetic binding leads to changes in protein structure and function.     

Molecular dynamics simulation of a synthetic four-alpha-helix bundle that binds the anesthetic halothane.

The structural features of binding sites for volatile anesthetics are examined by performing a molecular dynamics simulation study of the synthetic four-alpha-helix bundles (Aalpha2)2, which are formed by association of two 62-residue di-alpha-helical peptides. The peptide bundle (Aalpha2)2 was designed by Johansson et al. [Biochemistry 37 (1998) 1421-1429] and was shown experimentally to have a high affinity for the binding of the anesthetic halothane (CF3CBrCIH) in a hydrophobic cavity. Since (Aalpha2)2 can exhibit either the anti or syn topologies, the two distinct bundles are simulated both in the presence and in the absence of halothane. Nanosecond length molecular dynamics trajectories were generated for each system at room temperature (T = 298 K). The structural and dynamic effects of the inclusion of halothane are compared, illustrating that the structures are stable over the course of the simulation; that the (Aalpha2)2 bundles have suitable pockets that can accommodate halothane; that the halothane remains in the designed hydrophobic cavity in close proximity to the Trp residues with a preferred orientation; and that the dimensions of the peptide are perturbed by the inclusion of an anesthetic molecule.     

Effects of halothane, enflurane, and isoflurane on measurements of Ca(2+) by calcium electrode and aequorin luminescence

We assessed the possible effects of the volatile halogenated anesthetics halothane, enflurane, and isoflurane on Ca(2+) electrode measurements and on the Ca(2+) sensitivity of the bioluminescent protein aequorin. In Ca(2+)-EGTA buffers of different pCa values (7. 870, 6.726, 6.033, 4.974, 4.038, and 2.995) and in serial Ca(2+) dilutions (10(-4), 10(-3), and 10(-2) M), halothane, enflurane, and isoflurane each caused a concentration-dependent and reversible increase in the absolute value of the negative electrode potential. Isoflurane and enflurane had larger effects than halothane. Neither of these anesthetics changed aequorin luminescence at any pCa tested in the range 2-8. There was no potentiation or inactivation of aequorin luminescence over a period of up to 2 h. These results suggest that (1) halothane, enflurane, and isoflurane interfere with Ca(2+) electrode measurements, most likely by changing the physicochemical properties of the membrane; (2) these anesthetics do not inactivate or otherwise modify the characteristics of the reaction of Ca(2+) with aequorin; and (3) these anesthetics do not change the apparent affinity of EGTA for Ca(2+).     

Detergent-like action of the antibiotic peptide surfactin on lipid membranes

Surfactin is a bacterial lipopeptide with powerful surfactant-like properties. High-sensitivity isothermal titration calorimetry was used to study the self association and membrane partitioning of surfactin. The critical micellar concentration (CMC), was 7.5 microM, the heat of micellization was endothermic with DeltaH(w-->m)(Su) = +4.0 kcal/mol, and the free energy of micellization DeltaG(O,w-->m)(Su) = -9.3 kcal/mol (25 degrees C; 100 mM NaCl; 10 mM TRIS, 1 mM EDTA; pH 8.5). The specific heat capacity of micellization was deduced from temperature dependence of DeltaH(w-->m)(Su) as DeltaC(w-->m)(P) = -250 +/- 10 cal/(mol.K). The data can be explained by combining the hydrophobicity of the fatty acyl chain with that of the hydrophobic amino acids. The membrane partition equilibrium was studied using small (30 nm) and large (100 nm) unilamellar POPC vesicles. At 25 degrees C, the partition coefficient, K, was (2.2 +/- 0.2) x 10(4) M(-1) for large vesicles leading to a free energy of DeltaG(O, w-->b)(Su) = -8.3 kcal/mol. The partition enthalpy was again endothermic, with DeltaH(w-->b)(Su) = 9 +/- 1 kcal/mol. The strong preference of surfactin for micelle formation over membrane insertion explains the high membrane-destabilizing activity of the peptide. For surfactin and a variety of non-ionic detergents, the surfactant-to-lipid ratio, inducing membrane solubilization, R(sat)(b), can be predicted by the simple relationship R(sat)(b) approximately K. CMC.     

Partitioning of four modern volatile general anesthetics into solvents that model buried amino acid side-chains.

Partitioning of four modern inhalational anesthetics (halothane, isoflurane, enflurane, and sevoflurane) between the gas phase and nine organic solvents that model different amino acid side-chains and lipid membrane domains was performed in an effort to define which microenvironments present in proteins and lipid bilayers might be favored. Compared to a purely aliphatic environment (hexane), the presence of an aromatic-, alcohol-, thiol- or sulfide group on the solvent improved anesthetic partitioning, by factors of 1.3-5.2 for halothane, 1.7-5.6 for isoflurane, 1.7-7.6 for enflurane, and 1.5-7.3 for sevoflurane. The most favorable solvent for halothane partitioning was ethyl methyl sulfide, a model for methionine. Enflurane and isoflurane partitioned most extensively into methanol, a model for serine, and sevoflurane into ethanol, a model for threonine. Isoflurane also partitioned favorably into ethyl methyl sulfide. The results suggest that volatile general anesthetics interact better with partly polar groups, which are present on amino acids frequently found buried in the hydrophobic core of proteins, compared to purely aliphatic side-chains. Furthermore, if an anesthetic molecule was located in a saturated region of a phospholipid bilayer membrane, there would be an energetically favorable driving force for it to move into several higher dielectric microenvironments present on membrane proteins. The results provide evidence that proteins rather than lipids are the likely targets of volatile general anesthetics in biological membranes.     

Kinetic study of the thermal inactivation of cholinesterase enzymes immobilized in solid matrices

The thermal inactivation of immobilized cholinesterase enzymes (ChE) in solid matrices where the protein unfolding is blocked was studied, thus enabling investigation of the kinetics of the inactivation process directly from the native structure to the inactivated state. The thermal inactivation of butyrylcholinesterase (BChE), recombinant human acetylcholinesterase (rHuAChE), and eel acetylcholinesterase (AChE) enzymes was studied in dry films composed of poly(vinyl pyrollidone) (PVP), bovine serum albumin (BSA) and trehalose at 60 degrees -120 degrees C. The kinetics follows a bi-exponential decay equation representing a combination of fast and slow processes. The activation enthalpy DeltaH(#) and the activation entropy DeltaS(#) for each of the three enzymes have been evaluated. The values of DeltaH(#) for the fast process and for the slow process of BChE are 33+/-3, and 28+/-2 kcal/mol, respectively, and the values of DeltaS(#) are 0.84+/-0.04, and -18.2+/-0.5 cal/deg, respectively. The appropriate value of DeltaH(#) for rHuAChE is 26+/-2 Kcal/mol, for both processes and the values of DeltaS(#) are -17.6+/-0.9, and -23.0+/-0.9 cal/deg, respectively. Similarly, the values of DeltaH(#) for eelAChE are 30+/-3, 31+/-1 kcal/mol, and the values of DeltaS(#) are -6.7+/-0.5, -9.1+/-0.2 cal/deg respectively.     

The inhibition of calcium efflux from rat liver mitochondria by halogenated anesthetics

The halogenated anesthetics halothane, enflurane and isoflurane inhibit the calcium efflux induced by Ruthenium Red in isolated rat liver mitochondria. The extent of the inhibition is higher for enflurane (approximately 50%) than for either isoflurane (approximately 35%) or halothane (approximately 15%), and does not increase significantly between 0.1 and 0.6-1.0 mM anesthetic. Both the mitochondrial respiratory rate and transmembrane electrical potential are unaffected by the halogenated anesthetics concentrations capable to inhibit the efflux of calcium.     

Energetic basis of molecular recognition in a DNA aptamer

The thermal stability and ligand binding properties of the L-argininamide-binding DNA aptamer (5'-GATCGAAACGTAGCGCCTTCGATC-3') were studied by spectroscopic and calorimetric methods. Differential calorimetric studies showed that the uncomplexed aptamer melted in a two-state reaction with a melting temperature T(m)=50.2+/-0.2 degrees C and a folding enthalpy DeltaH(0)(fold)=-49.0+/-2.1 kcal mol(-1). These values agree with values of T(m)=49.6 degrees C and DeltaH(0)(fold)=-51.2 kcal mol(-1) predicted for a simple hairpin structure. Melting of the uncomplexed aptamer was dependent upon salt concentration, but independent of strand concentration. The T(m) of aptamer melting was found to increase as L-argininamide concentrations increased. Analysis of circular dichroism titration data using a single-site binding model resulted in the determination of a binding free energy DeltaG(0)(bind)=-5.1 kcal mol(-1). Isothermal titration calorimetry studies revealed an exothermic binding reaction with DeltaH(0)(bind)=-8.7 kcal mol(-1). Combination of enthalpy and free energy produce an unfavorable entropy of -TDeltaS(0)=+3.6 kcal mol(-1). A molar heat capacity change of -116 cal mol(-1) K(-1) was determined from calorimetric measurements at four temperatures over the range of 15-40 degrees C. Molecular dynamics simulations were used to explore the structures of the unligated and ligated aptamer structures. From the calculated changes in solvent accessible surface areas of these structures a molar heat capacity change of -125 cal mol(-1) K(-1) was calculated, a value in excellent agreement with the experimental value. The thermodynamic signature, along with the coupled CD spectral changes, suggest that the binding of L-argininamide to its DNA aptamer is an induced-fit process in which the binding of the ligand is thermodynamically coupled to a conformational ordering of the nucleic acid.     

Thermodynamic analysis of the binding of component enzymes in the assembly of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus

The peripheral subunit-binding domain (PSBD) of the dihydrolipoyl acetyltransferase (E2, EC 2.3.1.12) binds tightly but mutually exclusively to dihydrolipoyl dehydrogenase (E3, EC 1.8.1.4) and pyruvate decarboxylase (E1, EC 1.2.4.1) in the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus. Isothermal titration calorimetry (ITC) experiments demonstrated that the enthalpies of binding (DeltaH degrees ) of both E3 and E1 with the PSBD varied with salt concentration, temperature, pH, and buffer composition. There is little significant difference in the free energies of binding (DeltaG degrees = -12.6 kcal/mol for E3 and = -12.9 kcal/mol for E1 at pH 7.4 and 25 degrees C). However, the association with E3 was characterized by a small, unfavorable enthalpy change (DeltaH degrees = +2.2 kcal/mol) and a large, positive entropy change (TDeltaS degrees = +14.8 kcal/mol), whereas that with E1 was accompanied by a favorable enthalpy change (DeltaH degrees = -8.4 kcal/mol) and a less positive entropy change (TDeltaS degrees = +4.5 kcal/mol). Values of DeltaC(p) of -316 cal/molK and -470 cal/molK were obtained for the binding of E3 and E1, respectively. The value for E3 was not compatible with the DeltaC(p) calculated from the nonpolar surface area buried in the crystal structure of the E3-PSBD complex. In this instance, a large negative DeltaC(p) is not indicative of a classical hydrophobic interaction. In differential scanning calorimetry experiments, the midpoint melting temperature (T(m)) of E3 increased from 91 degrees C to 97.1 degrees C when it was bound to PSBD, and that of E1 increased from 65.2 degrees C to 70.0 degrees C. These high T(m) values eliminate unfolding as a major source of the anomalous DeltaC(p) effects at the temperatures (10-37 degrees C) used for the ITC experiments.     

Carbon monoxide production from five volatile anesthetics in dry sodalime in a patient model: halothane and sevoflurane do produce carbon monoxide; temperature is a poor predictor of carbon monoxide production

BACKGROUND: Desflurane and enflurane have been reported to produce substantial amounts of carbon monoxide (CO) in desiccated sodalime. Isoflurane is said to produce less CO and sevoflurane and halothane should produce no CO at all.The purpose of this study is to measure the maximum amounts of CO production for all modern volatile anesthetics, with completely dry sodalime. We also tried to establish a relationship between CO production and temperature increase inside the sodalime. METHODS: A patient model was simulated using a circle anesthesia system connected to an artificial lung. Completely desiccated sodalime (950 grams) was used in this system. A low flow anesthesia (500 ml/min) was maintained using nitrous oxide with desflurane, enflurane, isoflurane, halothane or sevoflurane. For immediate quantification of CO production a portable gas chromatograph was used. Temperature was measured within the sodalime container. RESULTS: Peak concentrations of CO were very high with desflurane and enflurane (14262 and 10654 ppm respectively). It was lower with isoflurane (2512 ppm). We also measured small concentrations of CO for sevoflurane and halothane. No significant temperature increases were detected with high CO productions. CONCLUSION: All modern volatile anesthetics produce CO in desiccated sodalime. Sodalime temperature increase is a poor predictor of CO production.     

Effects of isoflurane, halothane, and enflurane on myocardial flow and energy stores in the perfused rat heart

The effect of three volatile anesthetics (halothane, enflurane, and isoflurane) on coronary flow and metabolic state of isolated rat hearts was studied. These anesthetics are coronary dilators and their effects are dose dependent. At 2 MAC (minimum alveolar concentration), isoflurane, enflurane, and halothane increase coronary flow by 114 +/- 5.9, 93 +/- 6.1, and 77 +/- 6.4%, respectively (p less than 0.001). At these concentrations, they also have a modest but significant metabolic effect causing a 30% reduction in myocardial ATP and phosphocreatine levels, with no significant modification in ADP and AMP concentrations. Energy charge and lactate/pyruvate ratio were also unaffected by these anesthetics. The vascular and metabolic effects were reversible within 2 and 30 min, respectively. Perfusion of the hearts with a Krebs-Henseleit solution without Pi did not interfere with the vascular and the metabolic effect of the anesthetics; however, in this case, ATP and phosphocreatine concentration did not return to control levels after their discontinuation despite full recovery of the vascular effect. These data suggest that the volatile anesthetics have direct coronary vascular and myocardial metabolic effects and that these effects occur independently.     

Thermodynamic analysis of L-arginine and N omega-hydroxy-L-arginine binding to nitric oxide synthase

Isothermal titration calorimetry has been used to determine thermodynamic parameters of substrate binding to the oxygenase domain of neuronal nitric oxide synthase (nNOS(oxy)) in the presence of the cofactor tetrahydrobiopterin. The intermediate N(omega)-hydroxy-L-arginine (NHA) has a larger affinity than L-Arginine (L-Arg) for nNOS(oxy), with K(d)=0.4+/-0.1 microM and 1.7+/-0.3 microM at 25 degrees C, respectively. nNOS(oxy) binds NHA and L-Arg with DeltaH -4.1+/-0.2 and -1.0+/-0.1 kcal/mol and DeltaS=15 and 23 cal/Kmol respectively. NHA binding is more exothermic probably due to formation of an extra hydrogen bond in the active site compared to L-Arg. The changes in heat capacity (DeltaC(p)) are relatively small for binding of both NHA and L-Arg (-53+/-18 and -95+/-23 cal/L mol, respectively), which indicates that hydrophobic interactions contribute little to binding.     

Thermodynamics of nucleotide binding to NBS-I of the Bacillus subtilis preprotein translocase subunit SecA.

SecA is the dissociatable nucleotide and preprotein binding subunit of the bacterial translocase. The thermodynamics of nucleotide binding to soluble SecA at nucleotide binding site I were determined by isothermal titration calorimetry. Binding of ADP and non-hydrolyzable ATPgammaS is enthalpy-driven (DeltaH(0) of -14.44 and -5.56 kcal/mol, respectively), but is accompanied by opposite entropic contributions (DeltaS(0) of -18.25 and 9.55 cal/mol/K, respectively). ADP binding results in a large change in the heat capacity of SecA (DeltaC(p)=-780 cal/mol/K). It is suggested that ADP binding promotes the interaction between the two thermodynamically discernible domains of SecA which is accompanied by a shielding of hydrophobic surface from solvent.     

Cytochrome c peroxidase-cytochrome c complex: locating the second binding domain on cytochrome c peroxidase with site-directed mutagenesis

Cytochrome c peroxidase (CcP) can bind as many as two cytochrome c (Cc) molecules in an electrostatic complex. The location of the two binding domains on CcP has been probed by photoinduced interprotein electron transfer (ET) between zinc-substituted horse cytochrome c (ZnCc) and CcP with surface charge-reversal mutations and by isothermal titration calorimetry (ITC). These results, which are the first experimental evidence for the location of domain 2, indicate that the weak-binding domain includes residues 146-150 on CcP. CcP(E290K) has a charge-reversal mutation in the tight-binding domain, which should weaken binding, and it weakens the 1:1 complex; K(1) decreases 20-fold at 18 mM ionic strength. We have employed two mutations to probe the proposed location for the weak-binding domain on the CcP surface: (i) D148K, a "detrimental" mutation with a net (+2) change in the charge of CcP, and (ii) K149E, a "beneficial" mutation with a net (-2) change in the charge. The interactions between FeCc and CcP(WT and K149E) also have been studied with ITC. The CcP(D148K) mutation causes no substantial change in the 2:1 binding but an increase in the reactivity of the 2:1 complex. The latter can be interpreted as a long-range influence on the heme environment or, more likely, the enhancement of a minority subset of binding conformations with favorable pathways for ET. CcP(K149E) has a charge-reversal mutation in the weak-binding domain that produces a substantial increase in the 2:1 binding constant as measured by both quenching and ITC. For the 1:1 complex of CcP(WT), DeltaG(1) = -8.2 kcal/mol (K(1) = 1.3 x 10(6) M(-)(1)), DeltaH(1) = +2.7 kcal/mol, and DeltaS(1) = +37 cal/K.mol at 293 K; for the second binding stage, K(2) < 5 x 10(3) M(-)(1), but accurate thermodynamic parameters were not obtained. For the 1:1 complex of CcP(K149E), DeltaG(1) = -8.5 kcal/mol (K(1) = 2 x 10(6) M(-)(1)), DeltaH(1) = +2. 0 kcal/mol, and DeltaS(1) = +36 cal/K.mol; for the second stage, DeltaG(2) = -5.5 kcal/mol (K(1) = 1.3 x 10(4) M(-)(1)), DeltaH(2) = +2.9 kcal/mol, and DeltaS(2) = +29 cal/K.mol.     

Sevoflurane-induced structural changes in a four-alpha-helix bundle protein

The mechanisms whereby volatile general anesthetics reversibly alter protein function in the central nervous system remain obscure. Using three different spectroscopic approaches, evidence is presented that binding of the modern general anesthetic sevoflurane to the hydrophobic core of a model four-alpha-helix bundle protein results in structural changes. Aromatic residues in the hydrophobic core reorient into new environments upon anesthetic binding, and the protein as a whole becomes less dynamic and exhibits structural tightening. Comparable structural changes in the predicted in vivo protein targets, such as the gamma-aminobutyric acid type A receptor and the N-methyl-D-aspartate receptor, may underlie some, or all, of the behavioral effects of these widely used clinical agents.     

The peculiar nature of the guanidine hydrochloride-induced two-state denaturation of staphylococcal nuclease: a calorimetric study.

This work determines the ratio of DeltaH(vH) /DeltaH(cal) for staphylococcal nuclease (SN) denaturation in guanidine hydrochloride (GdnHCl) to test whether GdnHCl-induced denaturation is two-state. Heats of mixing of SN as a function of [GdnHCl] were determined at pH 7.0 and 25 degrees C. The resulting plot of DeltaH(mix) vs [GdnHCl] exhibits a sigmoid shaped curve with linear pre- and post-denaturational base lines. Extending the pre- and post-denaturational lines to zero [GdnHCl] gives a calorimetric DeltaH (DeltaH(cal)) of 24.1 +/- 1.0 kcal/mol, for SN denaturation in the limit of zero GdnHCl concentration. Guanidine hydrochloride-induced denaturation Gibbs energy changes in the limit of zero denaturant concentration (DeltaG degrees (N)(-)(D)) at pH 7. 0 were determined for SN from fluorescence measurements at fixed temperatures over the range from 15 to 35 degrees C. Analysis of the resulting temperature-dependent DeltaG degrees (N)(-)(D) data defines a van't Hoff denaturation enthalpy change (DeltaH(vH)) of 26. 4 +/- 2.8 kcal/mol. The model-dependent van't Hoff DeltaH(vH) divided by the model-independent DeltaH(cal) gives a ratio of 1.1 +/- 0.1 for DeltaH(vH)/DeltaH(cal), a result that rules out the presence of thermodynamically important intermediate states in the GdnHCl-induced denaturation of SN. The likelihood that GdnHCl-induced SN denaturation involves a special type of two-state denaturation, known as a variable two-state process, is discussed in terms of the thermodynamic implications of the process.