Here are several best practices for performing traditional binding experiments with the MicroCal PEAQ-ITC, VP-ITC and iTC200 systems. This is a continuation of previous blogs ‘Best Practices for Isothermal Titration Calorimetry to study binding interactions’ part 1, part 2 and part 3.

Common issues with ITC raw data, and possible causes

  • Large injection heats, due to large heats of dilution:

    • This is typically due to a mismatch of the macromolecule and ligand solutions, that is much larger than the actual binding heat.
    • The binding isotherm often looks like a straight line, with no evidence of binding saturation.
    • Typically, this large heat change cannot be corrected by the subtraction of a control titration.
  • DP does not return to pre-injection baseline before the next injection.

    • This can be due to insufficient time between injections to allow for complete measurement of binding heat.
    • Not all the heat change is measured per injection, affecting data quality. Adjust injection volume, reduce ligand concentration, and/or have a longer time between injections.
    • If you notice this effect during the experiment, you can increase the interval between injections for the remaining injections.
    • There can also be another (slower) process causing a heat change, such as conformational changes.
  • Spikes in injections, some peaks are much larger (or much smaller) than others:

    • Caused by bubbles in the ITC cell and/or syringe, or a dirty or damaged plunger tip in the ITC injector.
  • Noisy baseline and/or spikes between injections:

    • Air bubbles due to poor cell and/or syringe filling, dirty cells and/or syringe.
    • Also, can be due to a bent injection needle or an issue with the injection system.
  • Stepping baseline: there is a change in the heat capacity of the system, due to enzymatic hydrolysis, pH or buffer mismatch:

    • If studying binding between substrate and enzyme, use a non-hydrolyzable substrate.
    • Stepping baseline can also be due to bubbles in the ITC cell or syringe, due to under-filling.
  • No detectable binding heat:

    • There is no binding.
    • It is possible that binding is occurring, and the heat change is too low. The binding enthalpy can be lower than expected, and/or the binding affinity is weaker than expected.
    • To increase the heat change/injection, you need to increase the concentration of macromolecule and/or ligand; increase the injection volume, and/or change the experimental condition, such as the experimental temperature or buffer/pH.

Correcting for the heat of dilution, prior to data analysis

  • As discussed in previous blogs, close matching of the macromolecule and ligand solutions is essential to avoid large heats of dilution that could mask the actual binding signal. These non-binding related heats need to be subtracted from each of the points in the macromolecule-ligand titration binding isotherm before further data analysis. This can be accomplished in several ways:
  • An estimate of the heat of dilution can be obtained directly from the experimental titration by averaging the integrated heats associated with the final few injections. This subtraction can be done using the “Simple Math” option in Origin data analysis. This method assumes that the binding sites have been fully saturated with ligand by the end of the ITC experiment.
  • For PEAQ-ITC, one can also use the “fitted offset” option during data analysis for correcting for the heat of dilution.
    • If you have a control titration of ligand solution into buffer, you can perform a point-by-point subtraction of the integrated peak values from this control from those in the experiment. For small binding heats, this correction could result in a noisier binding isotherm, compared to subtracting a constant.
  • Note that the control titration should result in reproducible heats of injection throughout the titration and result in a linear plot with no or a shallow slope (Figure 1).
  • Instead of a point-by-point correction, you can create a line, or get a mean injection heat change for the control titration and subtract that from the experimental data.
  • If the final few injections of the experimental binding curve have a non-zero slope, and/or the control titration is non-linear or has a large slope, you should consider the possible reasons for such results and if that can be corrected for adequately.
  • After heats of dilution have been accounted for by one of these approaches, the enthalpy of binding at saturation should approach zero and the binding isotherm can be fit using an appropriate model to determine binding parameters.

Figure 1: Raw ITC data, with the control titration (ligand into buffer) (red), and the binding experiment (ligand into macromolecule) (black). Note that the peaks for the control titration are similar in shape and size to the final injections of the binding experiment.

Figure 1: Normalized heat changes, with the control titration (red), and the binding experiment (black). Data are fit to the one set of sites binding model.

Optimization of ITC experiments, after the first experiments

  • It is possible to shift and change the shape of the binding isotherm by adjusting the concentration of the cell sample and/or the syringe sample to optimize the quality of the data obtained. You can also change the injection volume and number of injections. If you have preliminary data, you can use the “Design Experiment” wizard advanced options, or another ITC data simulation tool, to simulate binding isotherms and design future experiment.
  • You may not be able to use the “optimal” concentration(s) for your binding: very low reactant concentrations might result in too small an experimental signal (which depends on ΔH of binding), while very high concentrations may simply be impractical or impossible to achieve for biopolymers (e.g. if a protein tends to aggregate at high concentration).
  • If you are not able to get the ligand concentration high enough to achieve binding saturation after injection of the contents of the syringe volume, you can keep the macromolecule-ligand complex in the ITC cell, fill the syringe with more ligand, and perform a second titration experiment. This can be repeated until binding saturation is reached.
    • To fit the data, use the Concatenation software tool to put together two (or more) ITC data files
    • Download MicroCal ITC concatenation software
  • What happens if the binding affinity and macromolecule concentration results in a C value outside of the optimal range? For very tight binding reactions (where conditions of C> 1000 are likely) only ΔH and N can be accurately determined directly from an ITC experiment. Additional approaches, such as using a displacement/competition experiment with a pre-bound weak-binding ligand, may however still allow the user to determine a KD.
  • For weak binding events it may still be possible obtain useful data working in the “low C range” (C values below 1-5) by using a very high concentration of ligand to drive binding to near saturation at the end of the titration. In this scenario, the binding isotherms tend to be non-sigmoidal, and may require fixing N during fitting, so only the binding affinity can be measured with confidence. ΔH will not be well defined by the experiment. However, in cases where N is known with confidence, and can be fixed in the fitting procedure if necessary, such experiments can still be very informative. You can also do a displacement/competitive experiment designed for characterizing weak binders.

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