Titration Calorimetry Standards and the Precision of Isothermal Titration Calorimetry Data

Current Isothermal Titration Calorimetry (ITC) data in the literature have relatively high errors in the measured enthalpies of protein-ligand binding reactions. There is a need for universal validation standards for titration calorimeters. Several inorganic salt co-precipitation and buffer protonation reactions have been suggested as possible enthalpy standards. The performances of several commercial calorimeters, including the VP-ITC, ITC200, and Nano ITC-III, were validated using these suggested standard reactions

Supramolecular Complexation of \u3cem\u3eN\u3c/em\u3e-Alkyl- and \u3cem\u3eN\u3c/em\u3e,\u3cem\u3eN\u3c/em\u3e′-Dialkylpiperazines with Cucurbit[6]uril in Aqueous Solution and in the Solid State

Water seeds: Complex stoichiometry/composition and degree of oligomerization (oligomeric supramolecular complex formation) of cucurbit[6]uril (CB[6]) with N-alkyl- and N,N′-dialkylpiperazine were investigated in aqueous solutions by means of isothermal titration calorimetry (ITC), ESI-MS, NMR and light scattering measurements. Complex stoichiometry/composition and degree of oligomerization (oligomeric supramolecular complex formation) of cucurbit[6]uril (CB[6]) with N-alkyl- and N,N′-dialkylpiperazine were investigated in aqueous solutions by means of isothermal titration calorimetry (ITC), ESI-MS, NMR and light scattering measurements. It was found that the complex stability and the degree of oligomerization increase with elongating the alkyl chain attached to the piperazine core. X-ray crystallographic studies revealed a clear correlation between the structure of CB[6]–alkylpiperazine crystals obtained from aqueous solutions and the molecular weight/properties of host–guest oligomers existed in the solution as supramolecular “seeds” of crystal formation

Analysis of the interaction of calcitriol with the disulfide isomerase ERp57

Calcitriol, the active form of vitamin D3, can regulate the gene expression through the binding to the nuclear receptor VDR, but it can also display nongenomic actions, acting through a membrane- associated receptor, which has been discovered as the disul de isomerase ERp57. The aim of our research is to identify the binding sites for calcitriol in ERp57 and to analyze their interaction. We rst studied the interaction through bioinformatics and uorimetric analyses. Subsequently, we focused on two protein mutants containing the predicted interaction domains with calcitriol: abb’- ERp57, containing the rst three domains, and a’-ERp57, the fourth domain only. To consolidate the achievements we used the calorimetric approach to the whole protein and its mutants. Our results allow us to hypothesize that the interaction with the a’ domain contributes to a greater extent than the other potential binding sites to the dissociation constant, calculated as a Kd of about 10−9 M

Physicochemical properties of nickel and cobalt sulphate solutions of hydrometallurgical relevance

Producing nickel and cobalt metal by high pressure acid leaching (HPAL) of nickel laterites is becoming one of Australia's largest mineral processing industries. However, the background chemical information for this process, including the fundamental physicochemical properties of acidic metal sulphate leachate solutions, is not well known. In order to improve the efficiency of current and future HPAL plants, high quality physicochemical and thermodynamic data will be necessary. This thesis reports measurements on the densities and heat capacities of nickel and cobalt sulphate solutions and their mixtures along with detailed studies of the nature of the species present and the thermodynamics of their interconversions. Densities and heat capacities of nickel and cobalt sulphate and perchlorate solutions and their ternary mixtures were measured using a vibrating tube densimeter and a flow microcalorimeter respectively. These data were used to calculate the apparent molal volumes and heat capacities of these solutions. Standard partial molal quantities were then obtained by appropriate extrapolation procedures, along with the volume and heat capacity changes of ion pair formation. A comparison has been made between experimental densities and heat capacities with those predicted by Young's rule. Good agreement was obtained except when the degree of complexation varied significantly in the mixtures. The various ion pair species in nickel and cobalt sulphate solutions, along with those of magnesium sulphate (which is a major impurity in HPAL leachates), were reinvestigated by dielectric relaxation spectroscopy. Doubly solvent separated ion pairs, solvent shared ion pairs and contact ion pairs were shown to exist simultaneously in solution and their concentrations were determined from dilute to near-saturated concentrations. Evidence for the possible existence of a triple ion, M2SO4 2+, was also obtained in highly concentrated solutions. The equilibrium constants of the stepwise reactions and the effective hydration numbers of ions and ion pairs were also calculated. The heats of complexation of nickel(II) and cobalt(II) sulphate were determined at different ionic strengths in sodium perchlorate media by titration calorimetry. These data were fitted to a specific ion interaction model to obtain the standard state values. The corresponding entropies of complexation were calculated and were found to be the major contributor to the stability of the complexes

Resolving Three-State Ligand-Binding Mechanisms By Isothermal Titration Calorimetry: A Simulation Study

In this paper, I theoretically analyzed ITC profiles for three-state equilibria involving ligand binding coupled to isomerization or dimerization transitions. Simulations demonstrate that the mechanisms where the free or ligand-bound protein undergoes dimerization (such that the ligand cannot bind to or dissociate from the dimer) produce very distinctive titration profiles. In contrast, profiles of the pre-existing equilibrium or induced-fit models cannot be distinguished from a simple two-state process, requiring data from additional techniques to positively identify these mechanisms

Interaction of Human Serum Albumin with short Polyelectrolytes: A study by Calorimetry and Computer Simulation

We present a comprehensive study of the interaction of human serum albumin (HSA) with poly(acrylic acid) (PAA; number average degree of polymerization: 25) in aqueous solution. The interaction of HSA with PAA is studied in dilute solution as the function of the concentration of added salt (20 - 100 mM) and temperature (25 - 37$^{\circ}$C). Isothermal titration calorimetry (ITC) is used to analyze the interaction and to determine the binding constant and related thermodynamic data. It is found that only one PAA chain is bound per HSA molecule. The free energy of binding $\Delta G_b$ increases with temperature significantly. $\Delta G_b$ decreases with increasing salt concentration and is dominated by entropic contributions due to the release of bound counterions. Coarse-grained Langevin computer simulations treating the counterions in an explicit manner are used study the process of binding in detail. These simulations demonstrate that the PAA chains are bound in the Sudlow II site of the HSA. Moreover, $\Delta G_b$ is calculated from the simulations and found to be in very good agreement with the measured data. The simulations demonstrate clearly that the driving force of binding is the release of counterions in full agreement with the ITC-data

Thermodynamics of the ATPase Cycle of GlcV, the Nucleotide-Binding Domain of the Glucose ABC Transporter of Sulfolobus solfataricus

ATP-binding cassette transporters drive the transport of substrates across the membrane by the hydrolysis of ATP. They typically have a conserved domain structure with two membrane-spanning domains that form the transport channel and two cytosolic nucleotide-binding domains (NBDs) that energize the transport reaction. Binding of ATP to the NBD monomer results in formation of a NBD dimer. Hydrolysis of the ATP drives the dissociation of the dimer. The thermodynamics of distinct steps in the ATPase cycle of GlcV, the NBD of the glucose ABC transporter of the extreme thermoacidophile Sulfolobus solfataricus, were studied by isothermal titration calorimetry using the wild-type protein and two mutants, which are arrested at different steps in the ATP hydrolytic cycle. The G144A mutant is unable to dimerize, while the E166A mutant is defective in dimer dissociation. The ATP, ADP, and AMP-PNP binding affinities, stoichiometries, and enthalpies of binding were determined at different temperatures. From these data, the thermodynamic parameters of nucleotide binding, NBD dimerization, and ATP hydrolysis were calculated. The data demonstrate that the ATP hydrolysis cycle of isolated NBDs consists of consecutive steps where only the final step of ADP release is energetically unfavorable.

Supramolecular assembly of cucurbit[6]uril and N-butyl-4-pyrrolidinopyridine

The nature of the supramolecular host-guest complex involving 4-pyrrolidinopyridine (BuPC4) and cucurbit[6]uril (Q[6]) has been investigated by NMR and UV spectroscopy, MALDI-TOF mass spectrometry, X-ray crystallography and isothermal titration calorimetry (ITC). The results revealed that the alkyl chain of the guest BuPC4 is located inside the cavity of the Q[6] host, whereas the other section of the BuPC4 guest remains outside of the portal