Calorimetry isothermal titration

The ITC instrument is very similar to the DSC apparatus described above. However, in this case, instead of changing the temperature, the sample (and reference) are kept at constant temperature (hence isothermal ). The sample cell is fitted with an injection syringe so that small amounts of another solution may be mixed with the sample. Typically this might be used to measure the heat of binding of a drug or inhibitor molecule to an enzyme. 

The different mixing vessels that are described in the previous paragraph are designed for the interaction between two components and are not well adapted for titration studies. In the Isothermal Titration Calorimetry, aliquots of sample B is added in a volume of sample A, located in the calorimetric vessel and maintained under a constant stirring (Fig. 2.26). 

Another sample C can also be added according to the reaction to be simulated. To allow such experimentations, the C80 calorimetric block is modified to adapt a magnetic stirring system at its bottom part. This leaves more space to adapt the different liquid and gas introduction lines. A heating cover has also to be used for the thermostatisation of the fluids at the temperature of the calorimeter. The liquids are introduced through a motor-driven syringe pump that allow a continuous or a step injection of defined volumes. 

Such a calorimetric technique is especially designed to follow the liquid adsorption of organic compounds on catalysts or zeolites [12, 21, 49]. The adsorption of n-butylamine (in decane) on a zeolite (in a solution of decane) illustrates the calori- 

Such a vessel is adapted to follow an organic reaction when several compounds 

It is also used to simulate the injection of very small amounts of water to produce a hydrolysis reaction (hydrolysis of ammonia borane for the production of hydrogen). 

ITC is a small-scale, small-sample-size, high-sensitivity version of the thermal titrimetry already discussed. The instrumentation consists of a highly sensitive calorimeter, capable of detecting heat effects as small as 100 nJ on 1 nmol or less of a biopolymer. Temperature stability is critical the TA Instruments Nano ITC provides temperature stability of 0.0002°C at 25°C, for example. Sample cells are made of Hastelloy or gold with a sample volume of 1-2 mL required, depending 

While ITC is particularly suitable to follow the energetics of an association reaction between biomolecules, the combination of ITC and DSC provides a more comprehensive description of 

I very slow on/slow off excluded from project 

Isothermal titration calorimetry (ITC) has gained much attention recently, as the application of thermodynamic data in the lead generation process has been proposed as a strategy both to improve the quality of lead series and to allow for a more effective lead optimization process [30-32]. The technique enables the determination of the affinity, the binding enthalpy, and the stoichiometry in a single experiment within a time frame of 1 h using as littie as 100 pg of a target protein. 

One prerequisite to get precise affinity values in ITC is related to the shape of the binding isotherm and thus the affinity of the compoimd. A typical experimental setup using 20 pM of a target protein would enable to determine accurate affinity values in the range of Ap = 5 pM to about 5 nM. If affinities are either lower (e.g., fragments or primary HTS hits) or higher (e.g., advanced compounds in lead identification and/or optimization), one has to use competition experiments, also referred to as displacement ITC [35]. In the case of weakly binding 

This is a powerful method of obtaining thermodynamic data such as A H and K, and hence A G and A S, and in addition the stoichiometry n of the reaction. The stoichiometry is 

Studies of the binding of olive oil phenolics to food proteins have been carried out [167]. These showed small heats of interaction, which precluded the calculation of the above thermodynamic parameters. The heat changes upon interaction of proteins with tannic acid were much larger and allowed the reliable calculation of K, n and AH (Table 9.10). 

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Table 5 Binding of palmatine to homo and hetero polymer of AT as revealed from isothermal titration calorimetry [176]...

Fig. 14 Isothermal titration calorimetry for the binding of berberine (a and d) and pal-matine (b and e) to tRNA. Reprinted from [214] with permission from Elsevier...

Alkaloids Spectrophotometry Spectrofluorimetry Isothermal titration calorimetry ... 

Ion transfer, 14 (1977) 1 Irinotecan, anticancer agent, 34 (1997) 68 Isothermal titration calorimetry, in drug design, 38 (2001) 309... 

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. 

T. K. Dam and C. F. Brewer, Thermodynamic studies of lectin-carbohydrate interactions by isothermal titration calorimetry, Chem. Rev., 102 (2002) 387 -29. 

Further experiments focused therefore on [RuCl(en)(r 6-tha)]+ (12) and [RuCl(rj6-p-cym)(en)]+ (22), which represent the two different classes, and their conformational distortion of short oligonucleotide duplexes. Chemical probes demonstrated that the induced distortion extended over at least seven base pairs for [RuCl(rj6-p-cym)(en)]+ (22), whereas the distortion was less extensive for [RuCl(en)(rj6-tha)]+ (12). Isothermal titration calorimetry also showed that the thermodynamic destabilization of the duplex was more pronounced for [RuCl(r 6-p-cym)(en)]+ (22) (89). DNA polymerization was markedly more strongly inhibited by the monofunctional Ru(II) adducts than by monofunctional Pt(II) compounds. The lack of recognition of the DNA monofunctional adducts by HMGB1, an interaction that shields cisplatin-DNA adducts from repair, points to a different mechanism of antitumor activity for the ruthenium-arenes. DNA repair activity by a repair-proficient HeLa cell-free extract (CFE) showed a considerably lower level of damage-induced DNA repair synthesis (about six times) for [RuCl(en)(rj6-tha)] + compared to cisplatin. This enhanced persistence of the adduct is consistent with the higher cytotoxicity of this compound (89). 

The principles of titration calorimetry will now be introduced using isoperibol continuous titration calorimetry as an example. These principles, with slight modifications, can be adapted to the incremental method and to techniques based on other types of calorimeters, such as heat flow isothermal titration calorimetry. This method, which has gained increasing importance, is covered in section 11.2. 

Another area where titration calorimetry has found intensive application, and where the importance of heat flow versus isoperibol calorimetry has been growing, is the energetics of metal-ligand complexation. Morss, Nash, and Ensor [225], for example, used potenciometric titrations and heat flow isothermal titration calorimetry to study the complexation of UO "1" and trivalent lanthanide cations by tetrahydrofuran-2,3,4,5-tetracarboxylic acid (THFTCA), in aqueous solution. Their general goal was to investigate the potential application of THFTCA for actinide and lanthanide separation, and nuclear fuels processing. The obtained results (table 11.1) indicated that the 1 1 complexes formed in the reaction (M = La, Nd, Eu, Dy, andTm)... 

E. Freire, O. L. Mayorga, M. Straume. Isothermal Titration Calorimetry. Anal. 

Christensen, T., B. Svensson, and B.W. Sigurskjold. 1999. Thermodynamics of reversible and irreversible unfolding and domain interactions of glucoamylase from Aspergillus niger studied by differential scanning and isothermal titration calorimetry. Biochemistry 38 6300-6310. 

Jelesarov, I. and H.R. Bosshard. 1999. Isothermal titration calorimetry and differential scanning calorimetry as complementary tools to investigate the energetics of biomolecular recognition. J Mol Recognit 12 3-18. 

Livingstone, J.R. 1996. Antibody characterization by isothermal titration calorimetry. Nature 384 491 192. 

Baker, B.M. and K.P. Murphy. 1996. Evaluation of linked protonation effects in protein binding reactions using isothermal titration calorimetry. Biophys J 71 2049-2055. 

Ac, acetyl AONs, antisense oligonucleotides B, boat Bn, benzyl Bz, benzoyl C, chair CD, circular dichroism CO, carbon monoxide ConA, concanavalin A DAST, diethylaminosulfur trifluoride DFT, density functional theory DMDO, dimethyldiox-irane DMT, dimethoxytriphenylmethyl DNA, deoxyribonucleic acid dsDNA, double-stranded DNA E, envelope Fmoc, fluorenylmethyloxycarbonyl GlcNAc, /V-acetylglucosamine ITC, isothermal titration calorimetry kcat, catalytic rate constant Aa, association constant K, inhibition constant KM, Michaelis constant LiSPh, lithium thiophenolate LPS, lipopolysaccharide pM, micromolar MMT,... 

Association Constants (K ) for Interaction Between ConA and Selected Monosaccharides by Isothermal Titration Calorimetry... 

The enthalpy change associated with formation of a thermodynamically ideal solution is equal to zero. Therefore any heat change measured in a mixing calorimetry experiment is a direct indicator of the interactions in the system (Prigogine and Defay, 1954). For a simple biopolymer solution, calorimetric measurements can be conveniently made using titra-tion/flow calorimeter equipment. For example, from isothermal titration calorimetry of solutions of bovine P-casein, Portnaya et al. (2006) have determined the association behaviour, the critical micelle concentration (CMC), and the enthalpy of (de)micellization. 

McClements, D.J. (2000). Isothermal titration calorimetry study of pectin-ionic surfactant interactions. Journal of Agricultural andFood Chemistry, 48, 5604-5611. 

Wangsakan, A., Chinachoti, P., McClements, D.J. (2001). Maltodextrin-anionic surfactant interactions isothermal titration calorimetry and surface tension study. Journal of Agricultural and Food Chemistry, 49, 5039-5045. 

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