Hoff Enthalpy Research Articles

Complexation of β‐lactam antibiotic drug carbenicillin to bovine serum albumin: Energetics and conformational studies

Binding of the antibiotic drug carbenicillin to bovine serum albumin (BSA) has been studied using isothermal titration calorimetry (ITC) in combination with fluorescence and circular dichroism (CD) spectroscopies. The thermodynamic parameters of binding have been evaluated as a function of temperature, ionic strength, and in the presence of anionic, cationic and nonionic surfactants, tetrabutylammonium bromide, and sucrose. The values of van't Hoff enthalpy do not agree with the calorimetric enthalpy indicating conformational changes in the protein upon drug binding. These observations are supported by the intrinsic fluorescence and CD spectroscopic measurements. A reduction in the binding affinity of carbenicillin to BSA is observed with increase in ionic strength of the solution, thereby suggesting, prevailing of electrostatic interactions in the binding process. The involvement of hydrophobic interactions in the binding of the drug to the protein is also indicated by a slight reduction in binding constant in the presence of tetrabutylammonium bromide. The experiments in the presence of sucrose suggest that hydrogen bonding is perhaps not dominant in the binding. The anionic surfactant sodium dodecyl sulphate (SDS) is observed to completely interfere in the ionic interactions in addition to its partial denaturing capacity. However, the presence of cationic surfactant hexadecyl trimethylammonium bromide (HTAB) and nonionic surfactant Triton-X 100 induce a slight reduction in the values of binding affinity. These calorimetric and spectroscopic results, provide quantitative information on the binding of carbenicillin to BSA and suggests that the binding is dominated by electrostatic interactions with contribution from hydrophobic interactions.

Equilibrium Unfolding Mechanism of Chicken Muscle Triose Phosphate Isomerase

Triose phosphate isomerase (TIM) was prepared and purified from chicken breast muscle. The equilibrium unfolding of TIM by urea was investigated by following the changes of intrinsic fluorescence and circular dichroism spectroscopy, and the equilibrium thermal unfolding by differential scanning calorimetry (DSC). Results show that the unfolding of TIM in urea is highly cooperative and no folding intermediate was detected in the experimental conditions used. The thermodynamic parameters of TIM during its urea induced unfolding were calculated as DeltaG degrees =3.54 kcal.mol(-1), and m(G) = 0.67 kcal.mol(-1)M(-1), which just reflect the unfolding of dissociated folded monomer to fully unfolded monomer transition, while the dissociation energy of folded dimer to folded monomer is probe silence. DSC results indicate that TIM unfolding follows an irreversible two-state step with a slow aggregation process. The cooperative unfolding ratio, DeltaH(cal)/DeltaH(vH), was measured close to 2, indicating that the two subunits of chicken muscle TIM unfold independently. The van't Hoff enthalpy, DeltaH(vH), was estimated as about 200 kcal.mol(-1). These results support the unfolding mechanism with a folded monomer formation before its tertiary structure and secondary structure unfolding.

Refinement of noncalorimetric determination of the change in heat capacity, ΔCp, of protein unfolding and validation across a wide temperature range

The change in heat capacity, DeltaC(p), on protein unfolding has been usually determined by calorimetry. A noncalorimetric method which employs the Gibbs-Helmholtz relationship to determine DeltaC(p) has seen some use. Generally, in this method the free energy change on unfolding of the protein is determined at a variety of temperatures and the temperature at which DeltaG is zero, T(m), and change in enthalpy at T(m) are determined by thermal denaturation and DeltaC(p) is then calculated using the Gibbs-Helmholtz equation. We show here that an abbreviated method with stability determinations at just two temperatures gives values of DeltaC(p) consistent with values from free energy change on unfolding determination at a much wider range of temperatures. Further, even the free energy change on unfolding from a single solvent denaturation at the proper temperature, when coupled with the melting temperature, T(m), and the van't Hoff enthalpy, DeltaH(vH), from a thermal denaturation, gives a reasonable estimate of DeltaC(p), albeit with greater uncertainty than solvent denaturations at two temperatures. We also find that nonlinear regression of the Gibbs-Helmholtz equation as a function of stability and temperature while simultaneously fitting DeltaC(p), T(m), and DeltaH(vH) gives values for the last two parameters that are in excellent agreement with experimental values.

Reversible Intramolecular Triplet−Triplet Energy Transfer in Benzophenone-N-methylphthalimide Dyad

In the present paper, we synthesized a series of benzophenone (BP)-N-methylphthalimide (MePI) dyads (Cn, n = 3, 6, and 9, where n denotes the number of methylene in the linker) and investigated the photochemical properties and intramolecular triplet-triplet energy transfer from BP(T1) to MePI. Formation of two different intramolecular complexes was found, that is, a ground-state complex and a singlet exciplex. The formation of the triplet-equilibrium between MeBP and MePI was observed. The triplet-equilibrium constant (1.0 and 1.1 for C6 and C9, respectively) and forward ((3.8 +/- 1.3) x 107 and (3.9 +/- 1.2) x 107 s-1 for C6 and C9, respectively) and back ((3.8 +/- 1.3) x 107 and (3.6 +/- 1.2) x 107 s-1 for C6 and C9, respectively) energy transfer rates were estimated from the result of transient absorption measurements. From the van't Hoff plots, enthalpy and entropy change for the equilibrium formation were estimated.

Lysozyme Thermal Denaturation and Self-Interaction: Four Integrated Thermodynamic Experiments for the Physical Chemistry Laboratory

As part of an effort to infuse our physical chemistry laboratory with biologically relevant, investigative experiments, we detail four integrated thermodynamic experiments that characterize the denaturation (or unfolding) and self-interaction of hen egg white lysozyme as a function of pH and ionic strength. Students first use Protein Explorer to examine the structure of lysozyme and its charge dependence on pH. Student groups are then assigned one of four 45 mM sodium acetate buffers: pH 3.6 with either 0 or 60 mM NaCl or pH 4.6 with either 0 or 60 mM NaCl. Using their assigned buffers, student groups determine the second virial coefficient of lysozyme using laser light scattering and gel permeation chromatography. Student groups also determine the van't Hoff enthalpy of unfolding by UV absorbance thermal denaturation and compare their results to the van't Hoff enthalpy and calorimetric enthalpy of unfolding determined by differential scanning calorimetry. We stress the use of complementary techniques to determine these thermodynamic variables for student validation of experimental results. Students present their results in a peer reviewed, journal-style report and, using the data from the entire class, must determine the dependence of the lysozyme second virial coefficient and unfolding enthalpies on pH and ionic strength. Upon completion of these experiments, students are anticipated to appreciate the pH dependence of protein charge and screening of protein electrostatic repulsion with ionic strength.

Thermal unfolding mechanism of lipocalin‐type prostaglandin D synthase

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is a dual-functioning protein in the lipocalin family, acting as a PGD(2)-synthesizing enzyme and as an extracellular transporter for small lipophilic molecules. We earlier reported that denaturant-induced unfolding of L-PGDS follows a four-state pathway, including an activity-enhanced state and an inactive intermediate state. In this study, we investigated the thermal unfolding mechanism of L-PGDS by using differential scanning calorimetry (DSC) and CD spectroscopy. DSC measurements revealed that the thermal unfolding of L-PGDS was a completely reversible process at pH 4.0. The DSC curves showed no concentration dependency, demonstrating that the thermal unfolding of L-PGDS involved neither intermolecular interaction nor aggregation. On the basis of a simple two-state unfolding mechanism, the ratio of van't Hoff enthalpy (DeltaH(vH)) to calorimetric enthalpy (DeltaH(cal)) was below 1, indicating the presence of an intermediate state (I) between the native state (N) and unfolded state (U). Then, statistical thermodynamic analyses of a three-state unfolding process were performed. The heat capacity curves fit well with a three-state process; and the estimated transition temperature (T(m)) and enthalpy change (DeltaH(cal)) of the N<-->I and I<-->U transitions were 48.2 degrees C and 190 kJ.mol(-1), and 60.3 degrees C and 144 kJ.mol(-1), respectively. Correspondingly, the thermal unfolding monitored by CD spectroscopy at 200, 235 and 290 nm revealed that L-PGDS unfolded through the intermediate state, where its main chain retained the characteristic beta-sheet structure without side-chain interactions.

Evaluation of conformation and association behavior of multivalent alanine-rich polypeptides.

Helical alanine-rich polypeptides with functional groups displayed along the backbone can display desired molecules such as saccharides or therapeutic molecules at a prescribed spacing. Because these polypeptides have promise for application as biomaterials, the conformation and association of these molecules have been investigated under biologically relevant conditions. Three polypeptide sequences, 17-H-3, 17-H-6, and 35-H-6, have been produced through recombinant techniques. Circular dichroic (CD) spectroscopy was used to monitor the secondary structure of the polypeptides in PBS (phosphate buffered saline, pH 7.4). The aggregation behavior in PBS was monitored via analytical ultracentrifugation and non-denaturing polyacrylamide gel electrophoresis. The three polypeptides adopt a highly helical structure at low and ambient temperatures, and when heated, undergo a helix-to-coil transition, typical of other alanine-rich peptide sequences. The melting temperatures and van't Hoff enthalpies, extracted from the CD data, suggest similar stability of the sequences. Although alanine-rich sequences can be prone to aggregation, there is no indication of aggregation for the three polypeptides at a range of concentrations relevant for possible biological applications. The helical polypeptides are monomeric under biologically relevant conditions enabling application of these polypeptides as useful scaffolds for ligand or drug display.

A spectral study of the interaction between chelerythrine chloride and cytidine

To study the possible anticancer mechanisms of chelerythrine (CHE), and its interactions with cytidine were investigated by UV–vis spectrophotometric and spectrofluorimetric measurements and by thermodynamic calculations. The binding of CHE to cytidine could be characterized by the hypochromic and bathochromic effects in the absorption bands, and the quenching of fluorescence intensity. The spectral data were fit by linear analysis, yielding a binding constant of 2.49 × 10 4 L mol −1at 25 °C of CHE and cytidine, and a van’t Hoff enthalpy of −20.02 kJ/mol for the exothermic interaction in the standard state. In addition, with Δ r G M Θ = − 25.09 kJ / mol and Δ r S m Θ = 17.01 J / mol K , the interactions should be entropy-driven.

A Spectral Study of the Interaction Between Chelerythrine Chloride and Adenosine

The possible anticancer mechanisms of chelerythrine (CHE) and its interactions with adenosine were investigated by UV‐visible spectrophotometric and spectrofluorimetric measurements and by thermodynamic calculations. The binding of CHE to adenosine could be characterized by the hypochromic and bathochromic effects in the absorption bands and the quenching of fluorescence intensity. The spectral data were fitted by linear analysis, yielding a binding constant of 8.68×104 L · mol−1 at 25°C of CHE with adenosine, and a van't Hoff enthalpy of 92.8 kJ/mol for the endothermic interactions. In addition, with ΔG=−28.2 kJ/mol and ΔS=406 J/mol · K, the interactions should be entropy‐driven.

Binding of bioactive phytochemical piperine with human serum albumin: A spectrofluorometric study

Piperine, the bioactive alkaloid compound of the spice black pepper (Piper nigrum) exhibits a wide range of beneficial physiological and pharmacological activities. Being essentially water-insoluble, piperine is presumed to be assisted by serum albumin for its transport in blood. In this study, the binding of piperine to serum albumin was examined by employing steady state and time resolved fluorescence techniques. Binding constant for the interaction of piperine with human serum albumin, which was invariant with temperature in the range of 17-47 degrees C, was found to be 0.5 x 10(5)M(-1), having stoichiometry of 1:1. At 27 degrees C, the van't Hoff enthalpy DeltaH degrees was zero; DeltaS degrees and DeltaG degrees were found to be 21.4 cal mol(-1) K(-1) and -6.42 kcal mol(-1). The binding constant increased with the increase of ionic strength from 0.1 to 1.0M of sodium chloride. The decrease of Stern-Volmer constant with increase of temperature suggested that the fluorescence quenching is static. Piperine fluorescence showed a blue shift upon binding to serum albumin, which reverted with the addition of ligands -triiodobenzoic acid and hemin. The distance between piperine and tryptophan after binding was found to be 2.79 nm by Förster type resonance energy transfer calculations. The steady state and time resolved fluorescence measurements suggest the binding of piperine to the subdomain IB of serum albumin. These observations are significant in understanding the transport of piperine in blood under physiological conditions.

Thermodynamic benchmark study using Biacore technology

A total of 22 individuals participated in this benchmark study to characterize the thermodynamics of small-molecule inhibitor–enzyme interactions using Biacore instruments. Participants were provided with reagents (the enzyme carbonic anhydrase II, which was immobilized onto the sensor surface, and four sulfonamide-based inhibitors) and were instructed to collect response data from 6 to 36 °C. van’t Hoff enthalpies and entropies were calculated from the temperature dependence of the binding constants. The equilibrium dissociation and thermodynamic constants determined from the Biacore analysis matched the values determined using isothermal titration calorimetry. These results demonstrate that immobilization of the enzyme onto the sensor surface did not alter the thermodynamics of these interactions. This benchmark study also provides insights into the opportunities and challenges in carrying out thermodynamic studies using optical biosensors.

A DSC study of zinc binding to bovine serum albumin (BSA)

The thermal denaturation of bovine serum albumin (BSA) is a kinetically and thermodynamically controlled process. The effects of zinc binding to bovine serum albumin (BSA), followed by differential scanning calorimetry (DSC), were investigated in this work, with the purpose of obtaining a better understanding of the albumin/zinc interaction. From the DSC curves, the thermodynamic parameters of protein denaturation were obtained, i.e., the temperature of thermal transition maximum (T m), calorimetric enthalpy (?Hcal), van't Hoff enthalpy (?HvH), the number of binding sites (I, II), the binding constants for each binding site (K bI, K bII) and the average number of ligands bound per mole of native protein X N. The thermodynamic data of protein unfolding showed that zinc binding to bovine serum albumin increases the stability of the protein (higher values of ?Hcal) and the different ratio ?Hcal/?HvH indicates the perturbation of the protein during thermal denaturation.

Using circular dichroism collected as a function of temperature to determine the thermodynamics of protein unfolding and binding interactions

Circular dichroism (CD) is an excellent spectroscopic technique for following the unfolding and folding of proteins as a function of temperature. One of its principal applications is to determine the effects of mutations and ligands on protein and polypeptide stability. If the change in CD as a function of temperature is reversible, analysis of the data may be used to determined the van't Hoff enthalpy and entropy of unfolding, the midpoint of the unfolding transition and the free energy of unfolding. Binding constants of protein-protein and protein-ligand interactions may also be estimated from the unfolding curves. Analysis of CD spectra obtained as a function of temperature is also useful to determine whether a protein has unfolding intermediates. Measurement of the spectra of five folded proteins and their unfolding curves at a single wavelength requires approximately 8 h.

Thermal denaturations of staphylococcal nuclease wild-type and mutants monitored by fluorescence and circular dichroism are similar: Lack of evidence for other than a two state thermal denaturation

It is unclear whether the thermal denaturation of staphylococcal nuclease is a two state, three state, or variable two state process. The thermal denaturation of wild-type staphylococcal nuclease was followed by tryptophan fluorescence and circular dichroism signal at 222 nm, forty-two and fourteen times, respectively. Analysis of this data using a simple two state model gave melting temperatures of 53.0 ± 0.4 °C (fluorescence) and 52.7 ± 0.6 °C (CD) and van't Hoff enthalpies of 82.4 ± 2.6 kcal/mol and 88.6 ± 4.2 kcal/mol. Ninety-seven mutants also had these parameters determined by both fluorescence and CD. The average difference between the melting temperatures was 1.05 ± 0.75° and the average difference between van't Hoff enthalpies was 1.6 ± 4.8 kcal/mol. These very similar results for the two spectroscopic probes of structure are discussed in the context of the different models that have been proposed for nuclease denaturation. It is concluded, for most nuclease variants, that the errors introduced by a two state assumption are negligible and either virtually all helical structure is lost in any initial unfolding event or any intermediate must have low stability.

Binding of Naproxen and Amitriptyline to Bovine Serum Albumin: Biophysical Aspects

Binding of the drugs naproxen (which is an anti-inflammatory) and amitriptyline (which is an anti-depressant) to bovine serum albumin (BSA) has been studied using isothermal titration calorimetry (ITC), in combination with fluorescence and circular dichroism spectroscopies. Naproxen is observed to bind more strongly to BSA than amitriptyline. The temperature-dependent ITC results indicate the interaction of one molecule of naproxen with more than one protein molecule. On the other hand, amitriptyline binds to BSA with a reaction stoichiometry that varies from 1:1.2 to 1:2.9. The van't Hoff enthalpy, which is calculated from the temperature dependence of the binding constant, agrees well with the calorimetric enthalpy in the case of naproxen binding to BSA, indicating adherence to a two-state binding process. However, their disagreement in the case of amitriptyline indicates conformational changes in the protein upon ligand binding, as well as with the rise in temperature. The spectroscopic results did not suggest appreciable conformational changes as a result of binding; hence, the discrepancy could be attributed to the temperature-induced conformational changes. With increases in the ionic strength, a reduction in the binding affinity of naproxen to BSA is observed. This suggests the prevailing electrostatic interactions in the complexation process. The preponderance of the hydrophobic interactions in the binding of amitriptyline to BSA is indicated by the absence of any dependence of the ionic strength. A predominance of electrostatic interactions in the case of naproxen binding to BSA and that of hydrophobic interactions in the case of amitriptyline binding to BSA is further strengthened by the results of the binding experiments performed in the presence of ionic and nonionic surfactants. The binding parameters indicate that Triton X-100 blocks the hydrophobic binding sites on BSA, thereby altering the binding affinity of amitriptyline toward BSA. A partial overlap of the binding sites for these drugs is indicated by the binding parameters obtained in the titration of naproxen to the amitriptyline-BSA complex and vice versa. Thus, the results provide a quantitative understanding of the binding of naproxen and amitriptyline to BSA, which is important in understanding their effect as therapeutic agents individually and in combination therapy.

Differential Distortion of Substrate Occurs When It Binds to DNA Photolyase: A 2-Aminopurine Study

Cyclobutylpyrimidine dimers (CPDs) are formed between adjacent pyrimidines in DNA when it is exposed to ultraviolet light. CPDs can be directly repaired by DNA photolyase (PL) upon absorption of blue-green light. We have used the fluorescent adenine analogue 2-aminopurine (2Ap) to probe the local double-helical structure of the DNA substrate when it binds to the protein. Duplex melting temperatures and van't Hoff enthalpies were obtained by both UV-vis absorption and fluorescence spectroscopies to ascertain the effect of the probe and CPD on DNA stability. Steady-state fluorescence measurements of the single- and double-stranded oligos showed that the local region around the 5'-side of the CPD lesion was more disrupted and destacked than the 3'-side in substrate-protein complexes. These results were compared with those of a protein-substrate crystal structure, demonstrating that the crystal structure and solution-state studies are in agreement with regard to the differential distortions of the target DNA at the active site of the protein.

Criteria for downhill protein folding: Calorimetry, chevron plot, kinetic relaxation, and single‐molecule radius of gyration in chain models with subdued degrees of cooperativity

Recent investigations of possible downhill folding of small proteins such as BBL have focused on the thermodynamics of non-two-state, "barrierless" folding/denaturation transitions. Downhill folding is noncooperative and thermodynamically "one-state," a phenomenon underpinned by a unimodal conformational distribution over chain properties such as enthalpy, hydrophobic exposure, and conformational dimension. In contrast, corresponding distributions for cooperative two-state folding are bimodal with well-separated population peaks. Using simplified atomic modeling of a three-helix bundle-in a scheme that accounts for hydrophobic interactions and hydrogen bonding-and coarse-grained C(alpha) models of four real proteins with various degrees of cooperativity, we evaluate the effectiveness of several observables at defining the underlying distribution. Bimodal distributions generally lead to sharper transitions, with a higher heat capacity peak at the transition midpoint, compared with unimodal distributions. However, the observation of a sigmoidal transition is not a reliable criterion for two-state behavior, and the heat capacity baselines, used to determine the van't Hoff and calorimetric enthalpies of the transition, can introduce ambiguity. Interestingly we find that, if the distribution of the single-molecule radius of gyration were available, it would permit discrimination between unimodal and bimodal underlying distributions. We investigate kinetic implications of thermodynamic noncooperativity using Langevin dynamics. Despite substantial chevron rollovers, the relaxation of the models considered is essentially single-exponential over an extended range of native stabilities. Consistent with experiments, significant deviations from single-exponential behavior occur only under strongly folding conditions.

Stability of DNA Duplexes Containing GG, CC, AA, and TT Mismatches

We employed salt-dependent differential scanning calorimetric measurements to characterize the stability of six oligomeric DNA duplexes (5'-GCCGGAXTGCCGG-3'/5'-CCGGCAYTCCGGC-3') that contain in the central XY position the GC, AT, GG, CC, AA, or TT base pair. The heat-induced helix-to-coil transitions of all the duplexes are associated with positive changes in heat capacity, DeltaC(p), ranging from 0.43 to 0.53 kcal/mol. Positive values of DeltaC(p) result in strong temperature dependences of changes in enthalpy, DeltaH degrees, and entropy, DeltaS degrees , accompanying duplex melting and cause melting free energies, DeltaG degrees, to exhibit characteristically curved shapes. These observations suggest that DeltaC(p) needs to be carefully taken into account when the parameters of duplex stability are extrapolated to temperatures distant from the transition temperature, T(M). Comparison of the calorimetric and van't Hoff enthalpies revealed that none of the duplexes studied in this work exhibits two-state melting. Within the context of the central AXT/TYA triplet, the thermal and thermodynamic stabilities of the duplexes in question change in the following order: GC > AT > GG > AA approximately TT > CC. Our estimates revealed that the thermodynamic impact of the GG, AA, and TT mismatches is confined within the central triplet. In contrast, the thermodynamic impact of the CC mismatch propagates into the adjacent helix domains and may involve 7-9 bp. We discuss implications of our results for understanding the origins of initial recognition of mismatched DNA sites by enzymes of the DNA repair machinery.

Thermodynamics of Interactions of TAlPyP4 and AgTAlPyP4 Porphyrins with Poly(rA)poly(rU) and Poly(rI)poly(rC) Duplexes

We employed UV light absorption and circular dichroism (CD) spectroscopic measurements to study the binding of novel water-soluble porphyrins meso-tetra-(4N-allylpyridyl)porphyrin [TAlPyP4], and its Ag containing derivative to the poly(rA)poly(rU) and poly(rI)poly(rC) RNA duplexes. Our results suggest that TAlPyP4 associate with the duplexes via intercalation, whereas the conservative CD spectra indicates that AgTAlPyP4 preferably binds via outside self-stacking mode. We used our determined binding isotherms for each ligand-RNA binding event to calculate the binding constant, Kb , and binding free energy, ΔGb = -RT1nKb. By performing these experiments as a function of temperature, we evaluated the van't Hoff binding enthalpies, ΔH. The binding entropies, ΔSb , were calculated as ΔSb = (ΔHb —ΔGb)/T. We interpret our data in terms of specific interactions that stabilize/destabilize each ligand- RNA complex studied in this work. Taken together, our data provide important new information about the thermodynamics of interactions of porphyrins with nucleic acids.

Solubility and Melting Properties of Salicylic Acid

The solubility of salicylic acid has been investigated in methanol, acetonitrile, acetic acid, acetone, water, and ethyl acetate from (10 to 50) °C. No new polymorphs or solvates of salicylic acid were found. The melting properties of salicylic acid were determined by differential scanning calorimetry. A correlation was observed between the solubility and the van't Hoff enthalpy of solution. A higher solubility was related to a lower van't Hoff enthalpy of solution. Water differed from the organic solvents in terms of solubility and its correlation to the van't Hoff enthalpy of solution. In addition, the morphology of salicylic acid crystals recrystallized from water differed from the other solvents.