MicroCal PEAQ-ITC Experiments – Practical Tips

Isothermal titration calorimetry (ITC) is one of the most robust and convenient techniques to thermodynamically examine intermolecular interactions by detecting any reaction heat that takes place in the ITC cell [1-9]. ITC provides beneficial information on binding affinity, binding stoichiometry and mechanisms, driving forces, (de)hydration, (de)protonation, interacting surface areas, interactions with salts, and molecular flexibility such as protein dynamics[1-8,10-15]. The ability of ITC to follow Michaelis-Menten kinetics [16], hydrolysis[17], and estimate the exchange rates (i.e., association and dissociation rates) of molecular interactions[18] has additionally extended the utility of ITC to a range of biological systems.

In the last 10 years, two ITC instruments, VP-ITC (MicroCal™, Malvern Panalytical, UK) and ITC200 (MicroCal™, Malvern Panalytical, UK) have noticeably contributed to thermodynamic studies on molecular associations. PEAQ-ITC (MicroCal™, Malvern Panalytical, UK) has recently appeared as a new model of the ITC instrument with improved sensitivity, a more convenient washing module, and convenient analysis software [19]. Thus, MicroCal PEAQ-ITC is also predicted to contribute to the understanding of binding energetics and extend the application of ITC.

So as to acquire accurate and precise ITC thermograms and data, a precise understanding of the procedures used for ITC experiments is needed. Several comprehensive manuals on PEAQ-ITC are extremely useful and available [20] for attaining information on how to effectively perform experiments and examine data. The following article will offer practical and key tips based on the author’s experience of ITC experiments for the better performance of PEAQ-ITC and its analysis that are not covered here.

 

 

 

Air Bubble Formation

A smaller cell volume in the PEAQ-ITC instrument (approximately 0.2 mL) than in the VP-ITC instrument (approximately 1.42 mL) is highly beneficial; it decreases the amount of sample and experimental time required and enables the measurement of heat variations that are not evidently observed using VP-ITC, such as the heat related to the dilution of salts at a high concentration (Figure 1).

Intermolecular interactions with low affinity need ITC titration to a high molar ratio, for which large quantities of sample should be used. Rapid equilibration and measurements maximize the number of ITC measurements for calculating standard deviations and the prospects to analyze various samples and conditions in a particular time period. These benefits are also highly effective in ITC studies using aggregation-prone samples as they reduce the time available for aggregation. Additionally, the smaller cell volume of the PEAQ-ITC instrument is practical for decreasing the potential nucleation of aggregation. However, a number of issues may be encountered when performing PEAQ-ITC with a smaller volume than VP-ITC, as explained below.

Heat of titration of high concentrations of NaCl monitored by different models of ITC instruments. ITC thermograms of titration of 1M NaCl in 10 M HCl solution in the ITC titration syringe to protein A in 10 M HCl solution in the ITC cell were recorded using the VP-ITC (left) and PEAQ-ITC instrument (right). ITC peaks after the final titration are shown in red and magnified in the inset for comparison. The molar concentration of NaCl after each titration of VP-ITC and PEAQITC measurements was the same. The titration volume was 2 µL for the first titration and 20 µL for the other titrations using the VP-ITC, and 0.8 µL for the first titration and 2.2 µL for the other titrations using the PEAQ-ITC

Figure 1. Heat of titration of high concentrations of NaCl monitored by different models of ITC instruments. ITC thermograms of titration of 1M NaCl in 10 M HCl solution in the ITC titration syringe to protein A in 10 M HCl solution in the ITC cell were recorded using the VP-ITC (left) and PEAQ-ITC instrument (right). ITC peaks after the final titration are shown in red and magnified in the inset for comparison. The molar concentration of NaCl after each titration of VP-ITC and PEAQITC measurements was the same. The titration volume was 2 µL for the first titration and 20 µL for the other titrations using the VP-ITC, and 0.8 µL for the first titration and 2.2 µL for the other titrations using the PEAQ-ITC

The Removal of Remaining Bubbles (ITC cell)

Data acquired in ITC experiments with the smaller cell volume of the PEAQITC instrument will be impacted more by the presence and development of bubbles in the cell, which can produce baseline spikes and low baselines, than will experiments using the VP-ITC instrument. Thus, clearance of bubbles from the cell is regularly performed. However, users may feel that it is not easy to load a small volume of solution into the PEAQ-ITC cell using a loading syringe to exclude bubbles, mainly those who are acquainted with VP-ITC, which has an approximately seven-fold larger cell volume.

Strong purging (i.e., rapid loading) can splatter the sample solution, causing sample loss. However, weak purging (i.e., low loading) will not successfully expel bubbles. Therefore, pumping of solution into the cell has to be performed as gently as possible without splattering the sample solution. Rotating the loading syringe as it is pulled out is also beneficial for avoiding bubbles. Extra lamps or lights help to detect bubbles, as light is not available in the cell unit.

Special care is encouraged when loading and purging colored samples, as colors, especially at relatively high concentrations, can hinder observations of bubbles, and also when bubble-forming samples such as surfactants are employed. Experiments using colored membrane proteins in a surfactant-containing solution are classic examples. A short period of centrifugation using a tabletop centrifuge and/or degassing is effective for eluding the generation of bubbles.

Repeating the insertion and removal of a titration syringe may be the last step to guaranteeing a bubble-free state of sample solution in the ITC cell. If one observes noisy ITC thermograms during equilibration or the early delay, stop the measurement and repeat the insertion and removal of a titration syringe again.

The Removal of Remaining Bubbles (ITC (Titration) Syringe)

The volume of the titration syringe of the PEAQ-ITC instrument (approximately 40 µL) is also smaller than that of the VP-ITC instrument (approximately 280 µL). Thus, bubbles remaining in the titration syringe of the PEAQ-ITC instrument may impact the precise and accurate volume of titration more than that of the VP-ITC. If bubbles are detected, press the 'Plunger/Refill' button in the instrument control bar placed at the bottom of the MicroCal PEAQ-ITC control software window and try to remove them. A short centrifugation and/or degassing are effective for avoiding the development of bubbles.

When bubbles persist, implement the following procedures by regulating several chief functions of the ITC titration syringe in the instrument control bar:

I. Press the 'Plunger Down' button to empty the titration syringe

II. Press the 'Load' button

Note 1: The 'Load' function is recommended as it is accompanied by degassing, which is effective for eliminating bubbles

Note 2: 'Open Fill Port' →'Plunger Down' is commonly effective before performing the 'Load' function

III. If bubbles are not removed, repeat procedures I and II.

IV. If the above approaches do not work, refill with sample which has undergone a short centrifugation with a benchtop centrifuge and/or degassing. Check the plunger tip and consider changing it after it has been used for over 200 - 300 ITC measurements.

If one is planning to use the same sample in the titration syringe for the subsequent ITC measurement, one may fill the titration syringe with the same sample without washing or rinsing the syringe (i.e., simply washing the cell). After rinsing the needle tip of the titration syringe with working solution and wiping it, press the 'Plunger Down' button and then the 'Load' button. This is an effective approach to load the sample without adding bubbles; however, caution is required for aggregation-prone samples.

Residual Solution in the ITC Cell

Users usually rinse the ITC cell using buffer solution, which does not comprise of analytes. This is a basic and crucial procedure for matching solution components between solutions in the cell and syringe, and therefore reducing background heat (i.e., control heat). However, it is hard in practice to eliminate all solution remaining in the cell using the loading syringe. The effects of remaining solution in the cell of the PEAQ-ITC instrument on variations in the concentration of analytes will be larger than those of the VP-ITC instrument, due to the smaller cell volume in the former, compared to the latter.

Analyzes using an inappropriate sample concentration will compromise the assessments of the affinity constant (association and dissociation constants), n-value, and other thermodynamic parameters, including variations in entropy (ΔS), enthalpy (ΔH), and Gibbs free energy (ΔG) with high accuracy and reproducibility. So as to minimize this problem, it is suggested that the cell is rinsed with working sample solution, following rinsing using analyte-free buffer solution.

Remaining Sample Solution in a Cell Reservoir

So as to load the sample solution and perform subsequent pumping to eliminate bubbles, insertion of the loading syringe into the cell is an imperative procedure that ejects sample solution corresponding to the volume of the needle of the loading syringe. If sample solution remains in a cell reservoir before taking out the loading syringe, the cell will be filled with sample solution.

Importance of Cleaning the Cell Reservoir and Pipette Retaining Nut

The systematic cleaning system in the PEAQ-ITC instrument, which mainly comprises of the cell unit, washing module, and controller PC, is highly efficient and robust. However, sticky samples may stay in the cell reservoir and bottom of the retaining nut of a pipette. Therefore, cleaning of the cell reservoir and the bottom of the retaining nut is advisable so as to ensure clean ITC thermograms and successful analyses (Figure 2).

Illustration of the retaining nut in the PEAQ-ITC pipette unit. The retaining nut is indicated with an arrow and label.

Figure 2. Illustration of the retaining nut in the PEAQ-ITC pipette unit. The retaining nut is indicated with an arrow and label.

Washing and Rinsing Error Troubleshooting

The PEAQ-ITC system is sensitive and intuitive. Therefore, it senses any inadequacies in the system and creates many case-sensitive messages for errors. When error messages occur regarding cleaning steps, judiciously verify the capping states of bottles, every connection of the fluid lines, the amounts of solution in each bottle (14% DECON or 20% Contrad™ 70, doubly deionized water (DDW), and methanol (HPLC grade or >99% pure), and fluid waste) in the washing module, and also the connections between the cell unit, washing module, and controller PC.

Miscellaneous Tips

  1. Recover residual sample from the titration syringe by clicking the 'Plunger Down' button in the instrument control bar, after rinsing the tip of the titration syringe using working solution.
  2. Rinse the tip of the titration syringe using working solution and wipe the tip as quickly and gently as possible, taking care not to damage the needle of the titration syringe prior to insertion of the ITC titration syringe into the ITC cell. Or else, titrants will react with analytes in advance of ITC experiments. This procedure becomes more critical when the concentrations of titrants are extremely high.
  3. Prior to refilling the reference cell with DDW, guarantee that the cell reservoir is clean, or else DDW in the reference cell may become polluted as DDW will overflow from the reference cell during refilling. Keeping the cell unit clean is a precondition of ITC.

References

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2. Pierce, M. M., Raman, C. S. and Nall, B. T. (1999) Isothermal titration calorimetry of protein-protein interactions. Methods 19, 213-221

3. Leavitt, S. and Freire, E. (2001) Direct measurement of protein binding energetics by isothermal titration calorimetry. Curr Opin Struct Biol 11, 560-566

4. Ladbury, J. E. and Williams, M. A. (2004) The extended interface: measuring non-local effects in biomolecular interactions. Curr Opin Struct Biol 14, 562-569

5. Velazquez Campoy, A. and Freire, E. (2005) ITC in the post-genomic era...? Priceless. Biophys Chem 115, 115-124

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7. Lee, Y. H., Ikegami, T., Standley, D. M., Sakurai, K., Hase, T. and Goto, Y. (2011) Binding energetics of ferredoxin-NADP+ reductase with ferredoxin and its relation to function. Chembiochem 12, 2062-2070

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9. Kim, J. Y., Kinoshita, M., Kume, S., Tomas, H. G., Sugiki, T., Ladbury, J. E., Kojima, C., Ikegami, T., Kurisu, G., Goto, Y., et al. (2016) Noncovalent forces tune the electron transfer complex between ferredoxin and sulfite reductase to optimize enzymatic activity. Biochem J 473, 3837-3854

10. Markova, N. and Hallen, D. (2004) The development of a continuous isothermal titration calorimetric method for equilibrium studies. Anal Biochem 331, 77-88

11. Olsson, T. S., Williams, M. A., Pitt, W. R. and Ladbury, J. E. (2008) The thermodynamics of protein-ligand interaction and solvation: insights for ligand design. J Mol Biol 384, 1002-1017

12. Fukuhara, A., Nakajima, H., Miyamoto, Y., Inoue, K., Kume, S., Lee, Y. H., Noda, M., Uchiyama, S., Shimamoto, S., Nishimura, S., et al. (2012) Drug delivery system for poorly water-soluble compounds using lipocalin-type prostaglandin D synthase. J Control Release 159, 143-150

13. Kume, S., Lee, Y. H., Miyamoto, Y., Fukada, H., Goto, Y. and Inui, T. (2012) Systematic interaction analysis of human lipocalin-type prostaglandin D synthase with small lipophilic ligands. Biochem J 446, 279-289

14. Kume, S., Lee, Y. H., Nakatsuji, M., Teraoka, Y., Yamaguchi, K., Goto, Y. and Inui, T. (2014) Fine-tuned broad binding capability of human lipocalin-type prostaglandin D synthase for various small lipophilic ligands. FEBS Lett 588, 962-969

15. Kinoshita, M., Kim, J. Y., Kume, S., Sakakibara, Y., Sugiki, T., Kojima, C., Kurisu, G., Ikegami, T., Hase, T., Kimata-Ariga, Y., et al. (2015) Physicochemical nature of interfaces controlling ferredoxin NADP(+) reductase activity through its interprotein interactions with ferredoxin. Biochim Biophys Acta 1847, 1200-1211

16. Todd, M. J. and Gomez, J. (2001) Enzyme kinetics determined using calorimetry: a general assay for enzyme activity? Anal Biochem 296, 179-187

17. Maximova, K. and Trylska, J. (2015) Kinetics of trypsin-catalyzed hydrolysis determined by isothermal titration calorimetry. Anal Biochem 486, 24-34

18. Vander Meulen, K. A., Horowitz, S., Trievel, R. C. and Butcher, S. E. (2016) Measuring the Kinetics of Molecular Association by Isothermal Titration Calorimetry. Methods Enzymol 567, 181-213

19. http://www.malvern.com/en/products/product-range/microcal-range/microcal-itc-range/microcal-peaq-itc-range/default.aspx

20. http://www.malvern.com/

This information has been sourced, reviewed and adapted from materials provided by Malvern Panalytical.

For more information on this source, please visit Malvern Panalytical.

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