Isothermal titration microcalorimetry

Isothermal titration calorimetry (ITC) has long been recognized as a useful tool for the evaluation of binding constants [51]. The field of calorimetry changed markedly during the early 1990s with the introduction of commercial titration microcalorimeters [52]. These devices, available from several suppliers, operate with volumes near one to two milliliters. Virtually all the data reported to date on the thermody- 

The entropy of binding is thus available by subtraction. The partial differential of enthalpy with respect to temperature defines the partial change in constant pressure heat capacity. Over short temperature ranges this derivative is approximated as 

---

T. K. Dam and C. F. Brewer, Multivalent protein-carbohydrate interactions Isothermal titration microcalorimetry studies, Methods Enzymol., 379 (2004) 107-128. 

Fig. 16 Heat generated (Q) as a function of the amount of Ag03SCp3 added to solutions of 36 (filled circle), 38 (filled diamond), and 37 (filled square) in THF, as determined by isothermal titration microcalorimetry (T=28 °C, c=0.2 mm)...

Frazier, R.A., Papadopoulou, A., Mueller-Harvey, I., Kissoon, D., and Green, R.J., Probing protein-tannin interactions by isothermal titration microcalorimetry, J. Agric. Food Chem., 51, 5189, 2003. 

Portnaya, I., Cogan, U., Livney, Y.D., Ramon, O., Shimoni, K., Rosenberg, M., Danino, D. (2006). Micellization of bovine p-casein studied by isothermal titration microcalorimetry and cryogenic transmission electron microscopy. Journal of Agricultural and Food Chemistry, 54, 5555-5561. 

Quantitative investigation of recognition of this pair of liposomes was performed with isothermal titration microcalorimetry (ITC). It has been found that one-to-one binding between adenine and barbituric acid in the lipid/water/lipid interface occurs. However at T= 58°C, above the main lipid phase transition, the situation is different and no liposomal binding is detected. This is mainly due to the molecular disorder within the bilayer (liquid-disordered/liquid ordered phase coexistence) that limits the capacity of complementary moieties to bind, due to the weakening of the hydrogen bonds at these high temperatures. 

Whereas the binding of CBMs to insoluble carbohydrates can be monitored by the UV absorption of the protein, relatively large amounts are required, and in some of the early studies on CBDs they were made radioactive with to increase sensitivity. The general technical problem of monitoring the binding of soluble, non-chromophoric ligands to soluble CBMs, has, however, been solved only in the last decade, with the advent of isothermal titration microcalorimetry... 

Co is the concentration defining the standard state by convention, Co = 1 mol. If Xd has been measured as a function of temperature, AH and A5d can be derived from its temperature dependence by applying the Vant Hoff law. In recent years, isothermal titration microcalorimetry (ITC) has also been used for that purpose. As reviewed by Fisher and Singh (1995) and Ladbury and Chowdhry (1996), microcalorimetry gives direct access to AH and, also, if the titration is performed at several temperatures, to the heat capacity change AGd at constant pressure. The first quantity is proportional to the heat evolved on mixing known amounts of the two components of a complex, and the latter is equal to... 

NB rrC = isothermal titration microcalorimetry. The enthalpy of ionization was plotted as a function of solution pH — DNA = titration performed in flie absence of DNA + DNA = titration performed in Oie presence of DNA complexed wiOi DOPE. 

Despite the great diversity in the design of microcalorimeters and the experimental procedures described in the literature [1-10], only two microcalorimetric methods have found widespread application in cydodextrin (CyD) studies and drug-design research. These two methods are differential scanning calorimetry (DSC) and isothermal titration microcalorimetry (ITC). DSC and ITC can be con-... 

The greatly improved sensitivity of the modem microcalorimeters for both differential scanning miCTOcalorimetry (DSC) and isothermal titration microcalorimetry (ITC) has made their application highly useful. Titration microcalorimetry yields binding constants and, when applied as a function of temperafiire, full thermodynamic details for the binding process. DSC provides details about the distmbance of the bilayer system by the solubilized guest from which conclusions may be drawn about the preferred binding locations. 

The tweezer-type receptor 43 that was developed by Kilbum and coworkers contains a disnbstitnted guanidinium group in the middle of the chain which was expected to bind to the terminal carboxylate gronp of peptidic guests (Scheme 22). The two peptide arms that are arranged in a parallel fashion serve to induce substrate selectivity. To test this idea, 43 was incubated in aqueous sodium borate buffer (pH 9.2, 16.7% DMSO) with a 1000-member library of tripeptides attached to a TentaGel resin via the amino terminus. Mainly hydrophobic amino acid residues were incorporated into these tripeptides to ensure that receptor substrate interactions are largely due to hydrophobic interactions. Receptor 43 was found to bind to about 3% of the library members and showed 95% selectivity for Val at the carboxylate terminus of the tripeptides and 40% selectivity for Glu(OfBu) at the amino terminus. A stability constant of 4 x lO M- was determined for the complex between 43 and Z-Glu(OrBu)-Ser(OfBu)-Val-0 in 16.7% DMSO/water (1 mM sodium borate buffer, pH 9.2) by means of isothermal titration microcalorimetry. 

Courtois J, Berret J-F (2010) Probing oppositely charged siufactant and copolymer interactions by isothermal titration microcalorimetry. Langmuir 26 11750-11758. doi 10.1021/ lal01475x... 

As it has been already mentioned, isothermal microcalorimeters are those calorimeters designed to work in the microwatt range under essentially isothermal conditions. Isothermal titration microcalorimetry (IT xC) is designed to connect extremely sensitive thermal measurement equipment (approx. 20-100 nanowatts) with an automatic syringe able to add reactants in successive injections to the solution with a precision of few nanoliters [32, 33]. Each injection produces specific heat effect, as shown in Fig. 10.5. The determination of heats evolved as a result of interaction between molecules is a main application of this variation of calorimetry. Consequently, isothermal titration calorimetry is a suitable method for studying degradations and biodegradations of versatile pollutants. 

Discussion of the effect of ligand structure on protein-carbohydrate affinity requires an evaluation of complex stability constants. A munber of biophysical techniques are appropriate for the study of protein-carbohydrate interaction many of the more enlightening strategies are the topics of separate chapters elsewhere in this volume. We describe below three techniques used extensively in glycobiology— inhibition of hemagglutination, enzyme-linked lectin assay (ELLA), and isothermal titration microcalorimetry—and we consider the types of information provided by each technique in order to facilitate appropriate interpretation of the data. 

---