Isothermal Titration Calorimetry (ITC)

The ITC experiments were performed using a VP-ITC instrument (MicroCal, GE Healthcare, Freiburg, Germany) which is controlled by the VPViewer software (MicroCal). The typical setup of an ITC instrument is schematically shown in Figure 6.3. The isothermal titration calorimeter consists of two identical cells which are composed of a highly efficient thermal conducting material and are enclosed by an adiabatic jacket. [312] The ITC unit directly measures the heat evolved or taken up in liquid samples as a result of mixing precise amounts of reactants. For injecting the reactant and subsequent mixing of the solution the instrument is equipped with a high-precision spinning syringe.

Sensitive thermocouple circuits are used to detect temperature differences between the reference cell, which is filled with water, and the sample cell. The latter is calibrated to power units and the direct observable signal is the time-dependent input of power in µcal sec^-1 which is needed to maintain temperature equilibrium. Therefore, a small constant amount of power is continuously applied to the offset heater of the reference cell throughout the ITC experiment. This causes the differential power (DP) feedback system to become positive to supply compensating power to the sample cell that will equilibrate the temperatures. During an exothermic reaction the temperature is increased in the sample cell. This causes a negative change from the DP feedback since less power is required to maintain equal temperatures between the cells. The opposite is true for endothermic reactions. The time integral of the peaks caused by injecting reactants into the sample cell then gives the thermal energy of the reaction.

The reference and sample cell of the VP-ITC instrument are composed of Hastelloy® Alloy C-276 which is a highly efficient thermal conductive material and is inert to many different solvents. The working volume of the sample cell is 1.4 mL and a total of 300 µl of reactant can be injected using the supplied spinning syringe. According to the manufacturer, the detection limit of the instrument is about 0.1 µcal. Thus, reliable measurements are performed when the recorded power is 0.1 µcal sec^-1 or higher.

Figure 6.3: Schematic diagram of an isothermal titration calorimeter. The instrument consists of a reference and a sample cell which are surrounded by an adiabatic outer shield (jacket). A high-precision spinning syringe titrates the ligand (e.g., the protein solution) into the sample cell filled with the macromolecule (e.g., the microgel dispersion). Temperature differences between the reference and sample cell caused by the injection of the ligand are sensed by thermocouples. Depending on the nature of the reaction (i.e., exothermic or endothermic) the feedback circuit will either increase or decrease the power applied to the sample cell in order to maintain the cells at an identical temperature. The heat flow per unit of time (dq/dt) is the observable signal in an ITC experiment.

To successfully set up an ITC experiment the concentration of the ligand and macromolecule have to be properly chosen. The optimal values for both concentrations, however, depend on the mechanism of binding as well as on the binding constant Kb. For a ligand which binds to a single set of independent binding sites the shape of the binding isotherm is strongly dependent on Kb. In particular, The shape of the binding isotherm changes according to the product c given by equation (6.8): [239,249]

𝑐𝑐=𝑁𝑁𝐾𝐾_b[𝑀𝑀]_t (6.8)

Figure 6.4 shows a set of simulated ITC titration curves for reactions of 1:1 stoichiometry and with different binding constants, i.e., varying values of c. [249] Accurate values for ΔHITC are determined from the early points of the titration curves with c > 50 because the reaction is near completion and ΔHITC is nearly independent of Kb. On the other hand, accurate fitting of the binding constant Kb

Figure 6.4: Simulated ITC titration curve for varying values of c and with N set to 1. Reprinted with permission from Turnbull, W. B.; Daranas, A. H. J. Am. Chem. Soc. 2003, 125, 14859-14866. Copyright © 2003, American Chemical Society.

requires c < 500. Under these conditions enough data points are measured to describe the curvature in the titration curve around the equivalence point. For values of c less than approximately 50, Kb and ΔHITC become interdependent and the fitting results are accompanied by large errors. Thus, only in the range between 50 and 500 the ITC isotherms can be deconvoluted to obtain accurate thermodynamic values. Consequently, the concentration of the carrier particle [M]t should be chosen properly in order to fulfil this requirement. By this means, Kb, N and ΔHITC can be determined with high precision from this fitting procedure.

Having optimised the concentrations of protein and microgel for the ITC analysis, the ITC experiments were conducted as follows:

Prior to the ITC experiment the protein and microgel solutions were stirred and degassed for 5 min at 1 K below the experimental temperature using the ThermoVac degassing and thermostatting station.

Degassing of the samples ensures bubble free loading of each and avoids the formation of bubbles during the ITC experiment. A total of 300 µl of the protein solution of given concentration was titrated into the sample cell filled with 1.4 mL of buffer-matched microgel dispersion using a stirring speed of 290 rpm. The reference power was set to 10 µcal sec^-1 for all experiments performed. The concentrations of protein and microgel used for the ITC experiments are listed in Table 6.6. During the course of the experiments the injection volume as well as the time interval between the injections was changed to allow for optimal data acquisition. During the first 20 injections the volume of the injected protein was set to 3 µL with an interval of 350 sec between each injection. For the subsequent 48 injections an injection volume of 5 µL and a spacing of 300 sec were chosen. The smaller injection volumes in the first part of the measurements lead to a high density of data points near the equivalence point where the slope and curvature of the binding isotherm is drastically altered. Control experiments were conducted by titrating the appropriate buffer solution into the microgel dispersion and by injecting the protein solution into the buffer, respectively.

After each experiment the sample cell and the syringe were cleaned using the ThermoVac apparatus.

The sample cell and auto-pipette are connected to the ThermoVac via plastic tubings and the cell cleaning apparatus. After removal of the sample solution from the sample cell, about 200 mL of detergent solution was passed through the syringe and sample cell and collected by the vacuum flask which was interposed between the sample cell and the ThermoVac system. Then the sample cell and

hydrogen peroxide solution was removed and the compartments were rinsed with another 100 mL of detergent solution and with about 800 mL of water. Finally, the cell and syringe were dried by using a small amount of acetone and by subsequent flushing with air.

Briefly, the acquired ITC data were analysed as follows: First, the baseline of the raw ITC data was set manually. Then the data points were integrated with respect to time and divided by the moles of injected protein. The integrated heats of the adsorption experiment were corrected for the integrated heat of dilution of the protein. Afterwards the ITC data were fitted using the supplied ITC module for ORIGIN 7.0 (MicroCal). The SSIS model was used to fit the adsorption data. A detailed description of the fitting procedure including the underlying equations is found in section 3.3.3.1.

Table 6.6: Experimental parameters of the thermodynamic analysis of protein adsorption performed by ITC.

System Buffer/ionic strength T[K] c(protein) [mM] c(microgel) [mM]

lysozyme/

6.5.3.1 Fluorescein Isothiocyanate (FITC)-Labelling of Lysozyme

Lysozyme was dissolved in 50 mM bicarbonate buffer pH 8.75 to a concentration of 10.02 g L^-1. Previous to the reaction, a fresh solution of 5 g L^-1 FITC in anhydrous dimethyl sulfoxide was prepared. An equimolar amount of FITC solution was added to the lysozyme solution and the mixture was allowed to stir for 1 h at room temperature. After this period of time the labelled lysozyme solution was adjusted to pH ≈ 2.0 using a 20 vol-% HCl solution and dialysed against 10 mM MOPS buffer solution pH 7.2 (Spectra Pore CE membrane, MWCO 1000) to remove excess FITC and to adjust the pH value and the ionic strength to the conditions used in the adsorption experiments. The degree of labelling and the concentration of lysozyme were determined by UV-vis spectroscopy (Agilent 8453, Agilent Technologies, Böblingen, Germany) at 293 K. Therefore the diluted solutions of FITC-labelled lysozyme (lysozyme^FITC) were subjected to UV-vis spectroscopy and the extinction