Impact of Titratable Groups in Studies with Isothermal Titration Calorimetry
Received Date: Dec 17, 2013 / Accepted Date: Dec 18, 2013 / Published Date: Dec 30, 2013
Isothermal Titration Calorimetry (ITC) is a commonly used technique to determine the stoichiometry, affinity, and enthalpy of binding reactions in solution [1,2]. In many cases, conclusions are made on the basis of titrations performed in one buffer system. In this article, we will demonstrate a potential problem that may lead to incorrect conclusions about the energetics of a given reaction based on data acquired using a single buffer system, which originates from changes in pKas of titratable groups upon complex formation.
In a binding reaction, pKas of titratable group on the protein and the ligand may shift upon formation of the complex. As a result, protons are either released or taken up by these groups and the buffer responds to such changes either by releasing or taking up protons to maintain pH. Therefore, the observed enthalpy (ΔHobs), includes contributions from the intrinsic enthalpy of binding (ΔHint) and the heat of ionization of the buffer (ΔHion). ΔHint can be determined based on the following equation and using different buffers with different ΔHion. While the y-intercept yields ΔHint, the slope of the line yields the net protonation, Δn. ΔHobs=ΔHint+Δn (ΔHion)
It is important to note, that the ΔHint, determined in this manner, will still include contributions from the heat of ionization of the titratable groups in the protein–ligand complex. Furthermore, it is also a common practice to include a certain level of salt (50-200 mM) to eliminate the heat of nonspecific binding of the buffer to the protein. The importance of determining the intrinsic enthalpy of a reaction can be seen in the experimental data shown in Figure 1. Titration of the aminoglycoside phosphotransferase(3′)-IIIa (APH) with the aminoglycoside antibiotic netilmicin yielded an endothermic reaction when the titration was performed in Tris-HCl pH 7.4 (Figure 1, right panel), while the same titration in PIPES buffer at the same pH yielded an exothermic reaction (Figure 1, left panel). Tris-HCl, ACES, and PIPES buffers have ΔHion of 11.7, 7.5, and 2.7 kcal/mol respectively . Analysis of these data (Table 1) as described above clearly shows that this is an exothermic reaction with the ΔHint=-7.7 ± 0.8 kcal/mol.
|Buffer||ΔHion (kcal/mol)||ΔHobs(kcal/mol)a||ΔHint (kcal/mol)||Δn|
|PIPES||2.7||-5.1 ± 0.04||-7.7 ± 0.8||0.82 ± 0.1|
|HEPES||5.0||-4.45 ± 0.05|
|ACES||7.4||-1.3 ± 0.08|
|Tris-HCl||11.7||1.6 ± 0.04|
All experiments were performed at 25°C. aGiven errors were calculated by fits to SEDPHAT . bErrors for ΔHint and Δn are derived from the linear regression line of ΔHobs vs. ΔHion plot
Table 1: Titration of APH with netilmicin in different buffers.
The ΔHint for this binding event is representative of a reaction with a favorable enthalpy, similar to the previously determined thermodynamic data for the binding of aminoglycosides to several enzymes that modify these antibiotics [4-10]. Data acquired in tris buffer alone would cause a misinterpretation of the binding of netilmicin to APH as an entropydriven complex formation and exception to observations with other aminoglycosides, which is obviously incorrect. Although binding of all aminoglycosides to aminoglycoside-modifying enzymes are accompanied by protonation/deprotonation of titratable groups (i.e., Δn ≠ 0), large negative enthalpy of binding was almost always larger than the positive contribution of the heat of buffer ionization and therefore all thermograms showed exothermic signal. However, when the binding enthalpy is small, as it was with netilmicin, the importance of the contribution of buffer becomes much more significant and may even lead to potential misinterpretations as demonstrated here.
As a final note, we should also mention that the value of Δn itself can also be misleading as it includes both the protonation and deprotonation of several functional groups and represents only the net protonation. In other words, conditions where Δn ≈ 0 do not guarantee the lack of protonation/deprotonation as shown earlier .
- Campoy AV, Freire E ( 2005) ITC in the post-genomic era...? Priceless. Biophys Chem 115: 115-124.
- Ladbury JE, Chowdry BZ (1996) Sensing the heat: the application of isothermal titration calorimetry to thermodynamic studies of biomolecular interactions. Chem Biol 3: 791-801.
- Goldberg RN, Kishore N, Lennen RM (2002) Thermodynamic quantities for the ionization reactions of buffers. Journal of Physical and Chemical Reference Data 31: 231-370.
- Schuck, P (2003) On the analysis of protein self-association by sedimentation velocity analytical ultracentrifugation. Anal Biochem 320: 104-124.
- Jing X, Wright E, Bible AN, Peterson CB, Alexandre G et al. (2012) Thermodynamic Characterization of a Thermostable Antibiotic Resistance Enzyme, the Aminoglycoside Nucleotidyitransferase (4 '). Biochemistry 51: 9147-9155.
- Norris AL, Ozen C, Serpersu EH (2010) Thermodynamics and Kinetics of Association of Antibiotics with the Aminoglycoside Acetyltransferase (3)-IIIb, a Resistance-Causing Enzyme. Biochemistry 49: 4027-4035.
- Norris AL, Serpersu EH (2013) Ligand promiscuity through the eyes of the aminoglycoside N3 acetyltransferase IIa. Protein Sci 22: 916-928.
- Ozen C, Serpersu EH (2004) Thermodynamics of aminoglycoside binding to aminoglycoside-3 '-phosphotransferase IIIa studied by isothermal titration calorimetry. Biochemistry 43: 14667-14675.
- Serpersu EH, Norris AL (2012) Effect of Protein Dynamics and Solvent in Ligand Recognition by Promiscuous Aminoglycoside-Modifying Enzymes. Advances in Carbohydrate Chemistry and Biochemistry, Academic Press, USA, 221-248.
- Wright E, Serpersu EH (2005) Enzyme-substrate interactions with an antibiotic resistance enzyme: Aminoglycoside nucleotidyltransferase(2 '')-Ia characterized by kinetic and thermodynamic methods. Biochemistry 44: 11581-11591.
- Ozen C, Malek JM, Serpersu EH (2006) Dissection of aminoglycoside-enzyme interactions: A calorimetric and NMR study of neomycin B binding to the aminoglycoside phosphotransferase(3 ')-IIIa. J AmChemical Soc 128: 15248-15254.
Citation: Rosendall KD, Serpersu EH (2013) Impact of Titratable Groups in Studies with Isothermal Titration Calorimetry. J Thermodyn Catal 5: e123. Doi: 10.4172/2157-7544.1000e123
Copyright: © 2013 Rosendall KD, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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