Isothermal titration calorimetry ITC

In titration calorimetry, the experimental procedure is usually as follows The cell (volume of ca. 1-2 mL) is filled with reactant A, and the other reactant B is injected into the solution in aliquots of 2-25 pL using a microliter syringe. Approximately 10-25 injections are usually perfonned with waiting times between injections of 5-20 min, depending on the kinetics of the reaction. The observed heat signals are then evaluated as a function of concentration of B in the cell or as a flinction of molar ratio B/A. These experiments can be performed in a temperature range between 2 and 80°C to determine the temperature dependence of the reaction enthalpy. 

Depending on the specific problems to be studied, it is often advantageous to perform also the reversed type of experiment, namely adding reactant A to a solution of B in the cell. Here, the heat of reaction is determined as a function of concentration of A. Both experiments are complementary and the data should be fitted with one and the same particular binding model. 

In many cases where the solubility of one of the components is very low, only one of the two experiments is possible, the solution of the reactant with the low solubility has to be filled into the cell, because otherwise the heats of reaction are too low to be detectable. 

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Instrumentation. H and NMR spectra were recorded on a Bruker AV 400 spectrometer (400.2 MHz for proton and 100.6 MHz for carbon) at 310 K. Chemical shifts (< are expressed in ppm coupling constants (J) in Hz. Deuterated DMSO and/or water were used as solvent chemical shift values are reported relative to residual signals (DMSO 5 = 2.50 for H and 5 = 39.5 for C). ESl-MS data were obtained on a VG Trio-2000 Fisons Instruments Mass Spectrometer with VG MassLynx software. Vers. 2.00 in CH3CN/H2O at 60°C. Isothermal titration calorimetry (ITC) experiments were conducted on a VP isothermal titration calorimeter from Microcal at 30°C. 

As observed in several of the examples above, designed receptors do not always give the results expected of them. In recent years, several investigations as to why this occurs have been undertaken. Some of these reports have been discussed briefly above, and here we will describe two more which utilize the powerful technique of isothermal titration calorimetry (ITC) to determine the thermodynamic driving forces for molecular recognition events. 

Isothermal titration calorimetry (ITC) dilution experiments were used to measure association constants and thermodynamic parameters for the formation of dimers 15-15 (cf. Section 14.09.3.1) <20010L3221>. Aggregates 15-15 are highly associated at 298K and entropically driven. The change in heat capacity (ACp) for the formation of dimer 15-15 was determined by ITC measurements from 288 to 328 K yielding the negative value (ACp = — 185 6 cal mol-1 K 1). It was concluded that the dimerization process is driven by hydrophobic effect. 

TRAP recruitment requires Hgand-induced changes in receptors that allow simultaneous interactions of each TRAP trimer with three receptor intracellular domains. This observation implicates that monomeric TRAF-receptor interactions are of low affinity so that the interactions do not occur in the absence of receptor activation. A number of quantitative biophysical characterizations with isothermal titration calorimetry (ITC) and surface plasma resonance (SPR) have provided solid support to this view (Table III). 

Calorimetry. Isothermal titration calorimetry (ITC) has recently been proposed as a general method for the determination of enzymatic reaction rates.20 This method is based on the relationship between the power needed to maintain constant temperature and the number of moles of substrate converted. Power, the measured quantity, is related to heat (Q) by differentiation ... 

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