Simultaneous determination of thermodynamic and kinetic data by isothermal titration calorimetry

BackgroundThermodynamic and binding kinetic data increasingly support and guide the drug optimization process.MethodsBecause ITC thermograms contain binding thermodynamic and kinetic information, an efficient protocol for the simultaneous extraction of thermodynamic and kinetic data for 1:1 protein ligand reactions from AFFINImeter kinITC in one single experiment are presented.ResultsThe effort to apply this protocol requires the same time as for the standard protocol but increases the precision of both thermodynamic and kinetic data.ConclusionsThe protocol enables reliable extraction of both thermodynamic and kinetic data for 1:1 protein-ligand binding reactions with improved precision compared to the ‘standard protocol’.General significanceThermodynamic and kinetic data are recorded under exactly the same conditions in solution without any labeling or immobilization from a protein sample that is not 100% active and would otherwise render the extraction of kinetic parameters impossible.     

Determination of thermodynamic functions from scanning calorimetry data

We present a theoretical basis and new methods for the determination of thermodynamic functions from scanning calorimetry data. A thermodynamic state is defined here as an ensemble of microstates in the system, and it can be defined only through assumptions of its heat capacity function and the two integral constants. With these assumptions, scanning calorimetry data can be analyzed using the single or double (or multi-) deconvolution presented here. New equations to calculate the van't Hoff enthalpy function and the calorimetric enthalpy function are presented. We prove that the agreement of these two functions is a necessary and sufficient factor for the condition that the system can be described with the assumed two-state model.     

Practical use of modified maximum likelihoods for stratified data

Stratified data arise in several settings, such as longitudinal studies or multicenter clinical trials. Between-strata heterogeneity is usually addressed by random effects models, but an alternative approach is given by fixed effects models, which treat the incidental nuisance parameters as fixed unknown quantities. This approach presents several advantages, like computational simplicity and robustness to confounding by strata. However, maximum likelihood estimates of the parameter of interest are typically affected by incidental parameter bias. A remedy to this is given by the elimination of stratum-specific parameters by exact or approximate conditioning. The latter solution is afforded by the modified profile likelihood, which is the method applied in this paper. The aim is to demonstrate how the theory of modified profile likelihoods provides convenient solutions to various inferential problems in this setting. Specific procedures are available for different kinds of response variables, and they are useful both for inferential purposes and as a diagnostic method for validating random effects models. Some examples with real data illustrate these points.     

Considering evolutionary processes in the use of single-locus genetic data for conservation,with examples from the Lepidoptera

The increasing popularity of molecular taxonomy will undoubtedly have a major impact on the practice of conservation biology. The appeal of such approaches is undeniable since they will clearly be an asset in rapid biological assessments of poorly known taxa or unexplored areas, and for discovery of cryptic biodiversity. However, as an approach for diagnosing units for conservation, some caution is warranted. The essential issue is that mitochondrial DNA variation is unlikely to be causally related to, and thus correlated with, ecologically important components of fitness. This is true for DNA barcoding, molecular taxonomy in general, or any technique that relies on variation at a single, presumed neutral locus. Given that natural selection operates on a time scale that is often much more rapid than the rates of mutation and allele frequency changes due to genetic drift, neutral genetic variation at a single locus can be a poor predictor of adaptive variation within or among species. Furthermore, reticulate processes, such as introgressive hybridization, may also constrain the utility of molecular taxonomy to accurately detect significant units for conservation. A survey of published genetic data from the Lepidoptera indicates that these problems may be more prevalent than previously suspected. Molecular approaches must be used with caution for conservation genetics which is best accomplished using large sample sizes over extensive geography in addition to data from multiple loci. Matthew L. Forister, Chris C. Nice and James A. Fordyce contributed equally to this paper.     

Estimating non-isothermal bacterial growth in foods from isothermal experimental data

AIM: To develop a mathematical method to estimate non-isothermal microbial growth curves in foods from experiments performed under isothermal conditions and demonstrate the method's applicability with published growth data. METHODS AND RESULTS: Published isothermal growth curves of Pseudomonas spp. in refrigerated fish at 0-8 degrees C and Escherichia coli 1952 in a nutritional broth at 27.6-36 degrees C were fitted with two different three-parameter 'primary models' and the temperature dependence of their parameters was fitted by ad hoc empirical 'secondary models'. These were used to generate non-isothermal growth curves by solving, numerically, a differential equation derived on the premise that the momentary non-isothermal growth rate is the isothermal rate at the momentary temperature, at a time that corresponds to the momentary growth level of the population. The predicted non-isothermal growth curves were in agreement with the reported experimental ones and, as expected, the quality of the predictions did not depend on the 'primary model' chosen for the calculation. CONCLUSIONS: A common type of sigmoid growth curve can be adequately described by three-parameter 'primary models'. At least in the two systems examined, these could be used to predict growth patterns under a variety of continuous and discontinuous non-isothermal temperature profiles. SIGNIFICANCE AND IMPACT OF THE STUDY: The described mathematical method whenever validated experimentally will enable the simulation of the microbial quality of stored and transported foods under a large variety of existing or contemplated commercial temperature histories.     

The feasibility of determining kinetic constants from isothermal titration calorimetry data

Isothermal titration calorimetry (ITC) has long been established as an excellent means to determine the thermodynamic parameters of biomolecular interactions. More recently, efforts have focused on exploiting the power/time trace (the “thermogram”) resulting from ITC experiments to glean kinetic association and dissociation rates for these interactions. The success of such analyses rests on the ability of algorithms to simulate with high accuracy the output of the calorimeter. Thus, several critical factors must be taken into account: the injection protocol, the kinetics of the interaction, accurate discovery of the instrumental response to heat signals, and the addition of unrelated signals. All of these aspects of extracting kinetic constants from thermograms have been considered and addressed in the current work. To validate the resultant methods, we performed several ITC experiments, titrating small-molecule inhibitors into solutions of bovine carbonic anhydrase II or titrating lysozyme into solutions of anti-lysozyme nanobodies. We found that our methods could arrive at kinetic constants that were close to the known values for these interactions taken from other methods. Finally, the effort to improve ITC kinetic characterizations uncovered a set of best practices for both the calorimetric experiment and the subsequent analyses (termed “kinetically optimized ITC” or “KO-ITC”) that is detailed in this work.     

Direct measurement of the thermodynamic parameters of amyloid formation by isothermal titration calorimetry

Amyloid fibril deposition is associated with over 20 degenerative diseases, including Alzheimer's, Parkinson's, and prion diseases. Although research over the last few years has revealed the morphology and structural features of the amyloid form, knowledge about the thermodynamics of amyloid formation is limited. Here, we report for the first time a direct thermodynamic study of amyloid formation using isothermal titration calorimetry. Beta(2)-microglobulin, a protein responsible for dialysis-related amyloidosis, was used for extending amyloid fibrils in a seed-controlled reaction in the cell of the calorimeter. We investigated the enthalpy and heat capacity changes of the reaction, where the monomeric, acid-denatured molecules adopt an ordered, cross-beta-sheet structure in the rigid amyloid fibrils. Despite the dramatic difference in morphology, beta(2)-microglobulin exhibited a similar heat capacity change upon amyloid formation to that of the folding to the native globular state, whereas the enthalpy change of the reaction proved to be markedly lower. In comparison with the native state, the results outline the important structural features of the amyloid fibrils: a similar extent of surface burial even with the supramolecular architecture of amyloid fibrils, a lower level of internal packing, and the possible presence of unfavorable side chain contributions.     

Quantum-chemical studies on thermodynamic feasibility of 1-methyl-2,4,5-trinitroimidazole processes

1-Methyl-2,4,5-trinitro imidazole (MTNI) is a well-known melt cast explosive possessing good thermal stability and impact insensitivity. MTNI has been synthesized from multi-step nitration followed by methylation of imidazole exhibiting low yield. It is desirable to screen the process thermodynamically for evaluating feasibility of the process. In the present investigations, B3LYP method in combination with 3-21G** basis set has been chosen to evaluate the enthalpy of formation for reaction species by designing reasonable isodesmic reactions. Thermodynamic feasibility of the processes has been worked out assuming free energies of reactions as derived from standard enthalpy and entropy of the reaction species. All possible synthesis routes for the target molecule, MTNI has been conceptualized from different precursors/intermediates viz. imidazole, 2-nitroimidazole, 4-nitroimidazole, 1-methyl imidazole and 2,4,5-triiodoimidazole. Various nitrating agents have been employed and their effect studied for deciding the feasibility of the reaction. It has been found that reaction entropy and enthalpy are favorable on usage of NO₂BF₄ as nitrating agent. The charge on nitro group (?Q_NO2) has been used for better understanding of the reactivity of substrates/intermediates. Overall, the study helped in screening several possible routes for MTNI synthesis and identified the thermodynamically feasible process by using NO₂BF₄ as nitrating agent.     

Calculating thermodynamic data for transitions of any molecularity from equilibrium melting curves

In this paper, we derive the general forms of the equations required to extract thermodynamic data from equilibrium transition curves on oligomeric and polymeric nucleic acids of any molecularity. Significantly, since the equations and protocols are general, they also can be used to characterize thermodynamically equilibrium processes in systems other than nucleic acids. We briefly review how the reduced forms of the general equations have been used by many investigators to evaluate mono- and bimolecular transitions, and then explain how these equations can be generalized to calculate thermodynamic parameters from common experimental observables for transitions of higher molecularities. We emphasize the strengths and weaknesses of each method of data analysis so that investigators can select the approach most appropriate for their experimental circumstances. We also describe how to analyze calorimetric heat capacity curves and noncalorimetric differentiated melting curves so as to extract both model-independent and model-dependent thermodynamic data for transitions of any molecularity. The general equations and methods of analysis described in this paper should be of particular interest to laboratories that currently are investigating association and dissociation processes in nucleic acids that exhibit molecularities greater than two.     

Simultaneous determination of thermodynamic and kinetic parameters of aminopolycarbonate complexes of cobalt(II) and nickel(II) based on isothermal titration calorimetry data

The influence of the different side chain residues on the thermodynamic and kinetic parameters for complexation reactions of the Co²⁺ and Ni²⁺ ions has been investigated by using the isothermal titration calorimetry (ITC) technique supported by potentiometric titration data. The study was concerned with the 2 common tripodal aminocarboxylate ligands, namely, nitrilotriacetic acid and N‐(2‐hydroxyethyl) iminodiacetic acid. Calorimetric measurements (ITC) were run in the 2‐(N‐morpholino)ethanesulfonic acid hydrate (2‐(N‐morpholino) ethanesulfonic acid), piperazine‐N ,N ′‐bis(2‐ethanesulfonic acid), and dimethylarsenic acid buffers (0.1 mol L⁻¹, pH 6) at 298.15 K. The quantification of the metal‐buffer interactions and their incorporation into the ITC data analysis enabled to obtain the pH‐independent and buffer‐independent thermodynamic parameters (K , ΔG , ΔH , and ΔS ) for the reactions under study. Furthermore, the kinITC method was applied to obtain kinetic information on complexation reactions from the ITC data. Correlations, based on kinetic and thermodynamic data, between the kinetics of formation of Co²⁺ and Ni²⁺ complexes and their thermodynamic stabilities are discussed.     

Effects of carbon and oxygen limitations and calcium concentrations on biofilm removal processes

Bacterial biofilm removal processes due to shear and catastrophic sloughing have been investigated in a turbulent flow system under conditions of carbon versus oxygen substrate limitations and varying aqueous phase calcium concentrations. Biofilm cellular and extracellular polymer carbon, total biofilm carbon and mass, and biofilm calcium concentrations are measured for pure culture biofilms of the facultative aerobe, Pseudomonas putida ATCC 11172. Results indicate oxygen-limited biofilms reach a higher steady-state biofilm organic carbon level than carbon-limited biofilms. Oxygen-limited biofilms also exhibit (1) a higher extracellular polymer-carbon: cell-carbon ratio throughout biofilm development and (2) a higher biofilm calcium content than carbon-limited biofilms. Increasing aqueous phase calcium concentrations increase the amount of biofilm calcium in both cases; the rate of calcium accumulation in oxygen-limited biofilms increases with increasing liquid phase calcium concentrations over the entire range studied while the rates of calcium accumulation in carbon-limited biofilms appear independent of aqueous phase calcium concentrations above 11.0 mg/L. Oxygen-limited biofilms with their higher extracellular polymer and calcium content exhibit shear removal rates that are 20-40% of those observed for carbon-limited biofilms. However, it is the oxygen-limited biofilms that experience catastrophic sloughing events. The carbon-limited biofilms studied here never sloughed even if subjected to intentional long-term deprivation of all nutrients. Reduced shear removal and the susceptibility to sloughing of the oxygen-limited biofilms are attributed to their more cohesive structure bought about by their relatively greater extracellular polymer production.     

Agricultural use of leachates obtained from two different vermicomposting processes

The objective of this paper was to investigate the possible agricultural use of the vermicomposting process leachates. Two leachates coming, respectively, from the vermicomposting of cow dung (SCD) and the vermicomposting of green forages (SGF), as well as solution of Hewitt (C) were used at a dose of 1 ml 500 ml(-1) in the foliar fertilization of tomato plants. Treatments were applied 30, 60 and 90 days after planting (DAP). The obtained results showed that foliar fertilization with SCD and SGF increased the morphological and chemical parameters on tomato crop with respect to the plants receiving foliar treatment with SH and C, possibly due to the humic substances content in SCD and SGF. The higher content of humic substances in SGF with respect to the SCD are possibly the responsible of the higher chlorophyll contents observed in the plants receiving the former treatment. This aspect possibly promoted an increase in plant photosynthesis and therefore an increase in fruit quality.     

A potential role for isothermal calorimetry in studies of the effects of thermodynamic non-ideality in enzyme-catalyzed reactions

Attention is drawn to the feasibility of using isothermal calorimetry for the characterization of enzyme reactions under conditions bearing greater relevance to the crowded biological environment, where kinetic parameters are likely to differ significantly from those obtained by classical enzyme kinetic studies in dilute solution. An outline of the application of isothermal calorimetry to the determination of enzyme kinetic parameters is followed by considerations of the nature and consequences of crowding effects in enzyme catalysis. Some of those effects of thermodynamic non-ideality are then illustrated by means of experimental results from calorimetric studies of the effect of molecular crowding on the kinetics of catalysis by rabbit muscle pyruvate kinase. This review concludes with a discussion of the potential of isothermal calorimetry for the experimental determination of kinetic parameters for enzymes either in biological environments or at least in media that should provide reasonable approximations of the crowded conditions encountered in vivo.