M2+â¢EDTA Binding Affinities: A Modern Experiment in

Laboratory Experiment pubs.acs.org/jchemeduc

M2+•EDTA Binding Affinities: A Modern Experiment in Thermodynamics for the Physical Chemistry Laboratory Leah C. O’Brien,*,† Hannah B. Root,† Chin-Chuan Wei,† Drake Jensen,† Nahid Shabestary,† Cristina De Meo,† and Douglas J. Eder‡ †

Department of Chemistry, Southern Illinois University Edwardsville, Edwardsville, Illinois 62026-1652, United States Emeritus, Southern Illinois University Edwardsville, Edwardsville Illinois 62026-2224, United States

‡

S Supporting Information *

ABSTRACT: Isothermal titration calorimetry was used to experimentally determine thermodynamic values for the ethylenediaminetetraacetic acid (EDTA)(aq) + M2+(aq) reactions (M2+ = Ca2+ and Mg2+). Students showed that for reactions in a N-(2-hydroxyethyl)piperazine-N′ethanesulfonic acid (HEPES) buffer (pH = 7.4), the Mg2+ + EDTA reaction was endothermic, while Ca2+ binding to EDTA was exothermic. EDTA is triply ionized at pH 7.4 and therefore must shed a proton to the buffer prior to chelating the M2+ ion; thus the observed reaction enthalpies are strongly dependent on pH and buffer ionization enthalpy.

KEYWORDS: Upper-Divion Undergraduate, Physical Chemistry, Aqueous Solution Chemistry, Biophysical Chemistry, Calorimetry/Thermochemistry, Metals, Thermodynamics, Hands-On Learning/Manipulatives, Laboratory Instruction

■

INTRODUCTION TO ISOTHERMAL TITRATION CALORIMETRY Isothermal titration calorimetry (ITC) is an experimental technique where the heat of reaction is measured for sequential injections of a titrant into the titrand contained within an adiabatic chamber. ITC was first described, to the best of our knowledge, in 1968 by H.D. Johnston.1,2 ITC gained in popularity in the 1970s, where cell volumes were on the order of 10−100 mL. In 1989, Wiseman et al.3 reported the development of an ultrasensitive titration calorimeter, and this instrument was the forerunner of the modern nano-ITCs designed for small-scale reactions with volumes ≤ 1 mL using mM and nM solutions. 4 Nano-ITC has found many applications in biochemistry and pharmaceutical research. Protein−protein, protein−drug, protein−DNA, and protein− ligand interactions are examples of intermolecular interactions that can be studied by ITC.3−14 Despite the explosion of nanoITC in physical biochemistry research and in the pharmaceutical industry in the past decade, only a few articles related to this important analytical method have appeared in the science education literature. Three papers from the Journal of Chemical Education15−17 were identified that describe ITC undergraduate experiments. The 2001 JCE paper15 describes an isothermal heat conduction calorimeter and offers several ideas for undergraduate experiments. The 2008 and 2011 JCE papers16,17 describe the construction of a low-cost, homemade isothermal calorimeter and a [Ba2+ + 18-crown-6] binding experiment. These experiments show the great variety in applications of © XXXX American Chemical Society and Division of Chemical Education, Inc.

calorimetry at the mL scale. However, the equipment described in these papers does not offer the ultrasensitivity of the modern nano-ITC systems that is needed for enzyme binding studies, which are normally conducted at the sub-mL scale. Schematic diagrams for the nano-ITC apparatus can be found online (e.g., see refs 18−21). Briefly, a reference cell and a sample cell are submerged in an adiabatic chamber, as shown in Figure 1. One reagent is contained in the sample cell, and the other reagent is loaded into a syringe injector. Both reagents are in an identical buffer solution to minimize mixing/dilution enthalpy. In a standard ITC experiment, the automatic injector introduces a known amount of solution into the sample cell using a programmable series of small injections. A sensitive thermocouple on each cell activates feedback electronics, which will increase or decrease the electrical current to the sample cell and maintain the cells at the exact same temperature difference. A graph of μW versus time produces the ITC isotherm. ΔH is obtained by integrating the area under the curve, and both exothermic and endothermic reactions can be monitored. Each titration peak is integrated separately, and these values are used to create the sigmoid-shaped curve of the integrated isotherm. The equilibrium binding constant K can be determined by the severity of the sigmoid curve, and the mole ratio n can be determined using the inflection point of the sigmoid curve. The experimentally determined ΔH° and K° from the ITC analyses can be used to calculate ΔG° and ΔS° based on the well-known

A

DOI: 10.1021/acs.jchemed.5b00159 J. Chem. Educ. XXXX, XXX, XXX−XXX

Journal of Chemical Education

Laboratory Experiment

Figure 1. ITC schematic (diagram courtesy of TA Instruments).

thermodynamic relationships, ΔG° = −RT ln(K°) = ΔH° − TΔS°. When the stoichiometry is well-known as in this example with 1:1 binding, n is the “site number” and is used as a float number to evaluate the acceptance of the data; a value that deviates significantly from 1 indicates problems in determining the sample concentrations. Nano-ITC has for the most part been utilized only in biophysical research laboratories due to, in part, the equipment expense (nano-ITC from TA Instruments > $70k) and the reagent costs (i.e., enzymes). However, the importance of hands-on experience with nano-ITC is growing since this is one of the few ways to study the weak biochemical interactions that often correlate with a biological response. Thus, experience with ITC is important for students seeking further research opportunities and employment in the broad fields of pharmaceutical and biochemistry. Associated with this work is a paper that describes a lysozyme•NAG3 binding experiment using competitive inhibition developed for the biochemistry laboratory at our institution.22 Because of the delicate nature of that experiment (e.g., enzyme costs, enzyme purification requirements, and careful microscale techniques), this more robust and inexpensive experiment (described in this manuscript) was also developed to be used as the initial introduction to ITC. Our work describes experiments using isothermal titration calorimetry to study the binding of ethylenediaminetetraacetic acid (EDTA) with M2+ cations, suitable for a one- or two-week experiment in an upper-level physical chemistry laboratory course.

acid, often abbreviated as EDTA or H4Y, is a useful chelating agent. EDTA has a high affinity for metal cations and binds to metal atoms in a hexadentate fashion with a 1:1 mol ratio. Students first prepared a 10.0 mM N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid (HEPES) buffer trimmed to pH = 7.4 and then made 0.80 mM Mg2+, 0.80 mM Ca2+, and 0.10 mM EDTA solutions using the buffer as solvent. The M2+ + EDTA reactions were conducted in a commercial NanoITC2G with a Hastelloy cell (TA Instruments, New Castle, DE) with a reaction cell of 964 μL and a 250 μL syringe. A blank run of M2+ into buffer only was run with both metals and used to correct for dilution enthalpy.

■

HAZARDS

There are no significant health hazards associated with this experiment. However, as with any chemical product, contact with unprotected bare skin; inhalation of vapor, mist, or dust in work place atmosphere; or ingestion in any form should be avoided by observing good laboratory practice. The solutions prepared in this lab are very dilute and nontoxic at these concentrations, yet they should be handled with care and disposed of properly. Eye protection should be worn throughout the experiment. Particular care should be taken when handling the long, blunt-tip syringes used to fill the sample cell of the ITC. They should be held in a vertical position with the tip down and used only in the immediate vicinity of the ITC. The titration syringe used in the buret has a stir-paddle at the tip and thus poses no threat of puncture. However, the titration syringes are delicate and expensive; care must be taken to avoid bending the titration syringes tips.

■

EXPERIMENT This experiment studied the [M2+ + EDTA] reaction for M2+ = Ca2+ and Mg2+ using ITC at 25 °C. Ethylenediaminetetraacetic B




Figure 2. Isotherms (top) and integrated isotherm (bottom) for [EDTA + M2+] binding by ITC, where M2+ = Ca2+ (left) and M2+ = Mg2+ (right); 0.80 mM M2+ (titrant), 0.10 mM EDTA (titrand) buffered in HEPES (pH = 7.4); one injection of 10 μL followed by 12 injections of 20 μL each. The y-axis of the isotherm (top) indicates the differential power applied between the sample cell and the reference cell such that both cells are maintained at 25 °C, and an exothermic event is shown as an upward peak. The last few peaks in each isotherm can provide estimates for the enthalpy of dilution of the M2+ solution and any buffer mis-match between the titrant and titrand. These contributions mitigated by subtracting the isotherm from a blank run from the isotherm of an experimental run, where the blank is the titration of M2+ into a buffer only titrand (no EDTA).

Table 1. Thermodynamic Properties for [Mg2+ + EDTA] and [Ca2+ + EDTA] Binding Obtained from Student Experiments Compared with Literature Valuesab Experimental Conditions

Source

K (106 M−1)

ΔH (kJ/mol)

n

Buffer Ionization Enthalpy (kJ/mol)bg

Mg2+ + EDTA in HEPES,c pH = 7.4

Student lab results Literature valuea Student lab results Literature valuea Literature valueb Literature valueb Literature valueb

1.06 0.60 37.9 44 198 235 104

+18.7 +18.4 −21.7 −25.0 −50.0 −24.0 −13.9

0.93 1.0 1.01 1.0 1.0 0.99 1.01

21.7

Ca2+ + EDTA in HEPES,c pH = 7.4 Ca2+ + EDTA in TRIS,d pH = 7.5 Ca2+ + EDTA in MOPS,e pH = 7.5 Ca2+ + EDTA in PIPES,f pH = 7.5

21.7 47.4 21.9 11.5

a

Ref 6. bRef 5. cHEPES is 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid. dTRIS is tris(hydroxymethyl)aminomethane. eMOPS is 3-(Nmorpholino)propanesulfonic acid. fPIPES is piperazine-N,N′-bis(2-ethanesulfonic acid). gRef 23.

■

RESULTS AND DISCUSSION Representative isotherms and integrated isotherms from student work are shown in Figure 2. The thermodynamic values obtained from the experiments are given in Table 1 and are compared with literature values.5,6 Students found that [Ca2+ + EDTA] binding is an exothermic process, whereas [Mg2+ + EDTA] binding was an endothermic process. The laboratory handout developed for our students is available in the Supporting Information. The handout includes instruction on careful handling and loading of the ITC and gives suggestions for the preparation of quantitatively accurate,

dilute solutions. The handout also provides the pKa,n values for HEPES (n = 1, 2) and EDTA (n = 1−6), as well as a brief introduction to ITC, with example calculations for molecular concentrations (EDTA, M2+, and EDTA:M2+ complex) before and after a single injection. Time required for solution preparation, adjusting pH, and degassing is on the order of 40 min. A typical ITC run is approximately 2.5 h, where the first hour is temperature equilibration of the apparatus. During ITC run time, students use an ITC simulation program included in NanoAnalyze24 provided by TA Instruments to explore reaction characteristics and experimental C



■

conditions necessary for successful ITC experiments. For example, reagent concentrations must be chosen so that (i) the total heat of the reaction is readily observable with an acceptable S/N ratio and (ii) there is adequate conversion of titrand to product to ensure that the equilibrium constant K is well-determined.25 Both of the commercial ITC vendors include a simulation program with their software package, and a third simulation program is described (and available) in the literature.25 EDTA binds more strongly to Ca2+ than Mg2+ due to the favorable size match between the EDTA and Ca2+ ionic radius, and most students expected different binding enthalpies. However, all of the students (to date) have been surprised by the endothermic reaction of [Mg2+ + EDTA] in the HEPES buffer at pH = 7.4. A series of postlab discussion questions helped to guide students through the reaction for a better understanding of their results. These included, “Identify the conjugate acid/base pair for the HEPES buffer at pH = 7.4”, “Identify the exact chemical formula of EDTA at this pH”, “Identify all bonds that are broken and all bonds that are formed during the reaction”, and “Where does the proton from the EDTA go?”. At a pH of 7.4, all carboxyl groups are deprotonated to carboxylate groups, one amino nitrogen with pKa,5 = 6.16 is neutral (not protonated), while the other amino group with pKa,6 = 10.3 is protonated, leaving EDTA (pH = 7.4) triply ionized as HY3−. EDTA therefore must shed a proton to the buffer prior to chelating metal cations and creating the [M2+•Y4−]2− complex. Thus, the observed binding enthalpies are strongly dependent on pH and buffer ionization enthalpy.5,23,26 Assessment results of student laboratory reports are described in the Supporting Information. In summary, the isothermal calorimetry experiment, when paired with inferential questions, brought about observable, effective critical thinking among students. It appears that students were more successful in thinking linearly about direct causes and consequences but less successful in thinking broadly (suppose conditions were altered, what then?).

■


ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available on the ACS Publications website at DOI: 10.1021/acs.jchemed.5b00159. Detailed student handout (PDF, DOCX) Detailed instructor handout (PDF, DOCX) Discussion of educational goals, assessment of student laboratory reports (PDF, DOCX)

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We thank the students of Physical Chemistry Laboratory at Southern Illinois University Edwardsville (Summer 2010, Fall 2010, Summer 2011, Fall 2011) for their efforts in conducting experiments and providing instructive feedback on the student handout. This work was sponsored by the National Science Foundation Centers for Chemical Innovation (NSF-CCI) program (Award No. DUE-0941517).

■

REFERENCES

(1) Johnston, H. D. Thermodynamics (log K, ΔH°, ΔS°, ΔCP°) of Metal Ligand Interaction in Aqueous Solution. Design and Construction of an Isothermal Titration Calorimeter. Interaction of Cyanide Ion with Ni, Zn, Cd, and Hg. Interaction of Glycinate Ion with Mn, Fe, Co, Ni, Cu, Zn, and Cd. Ph.D. Dissertation, Brigham Young University, 1968. (2) Christensen, J. J.; Johnston, H. D.; Izatt, R. M. Isothermal titration calorimeter. Rev. Sci. Instrum. 1968, 39 (9), 1356−1359. (3) Wiseman, T.; Williston, S.; Brandts, J. F.; Lin, L. N. Rapid measurement of binding constants and heats of binding using a new titration calorimeter. Anal. Biochem. 1989, 179 (1), 131−137. (4) Plotnikov, V. V.; Brandts, J. M.; Lin, L.-N.; Brandts, J. F. A new ultrasensitive scanning calorimeter. Anal. Biochem. 1997, 250 (2), 237−244. (5) Griko, Y. V. Energetics of Ca2+−EDTA interactions: Calorimetric study. Biophys. Chem. 1999, 79 (2), 117−127. (6) Henzl, M. T.; Larson, J. D.; Agah, S. Estimation of parvalbumin Ca2+- and Mg2+-binding constants by global least-squares analysis of isothermal titration calorimetry data. Anal. Biochem. 2003, 319 (2), 216−233. (7) José, T. J.; Conlan, L. H.; Dupureur, C. M. Quantitative evaluation of metal ion binding to PvuII restriction endonuclease. JBIC, J. Biol. Inorg. Chem. 1999, 4 (6), 814−823. (8) Lopez, M. M.; Chin, D.-H.; Baldwin, R. L.; Makhatadze, G. I. The enthalpy of the alanine peptide helix measured by isothermal titration calorimetry using metal-binding to induce helix formation. Proc. Natl. Acad. Sci. U. S. A. 2002, 99 (3), 1298−1302. (9) Gourishankar, A.; Shukla, S.; Ganesh, K. N.; Sastry, M. Isothermal Titration Calorimetry Studies on the Binding of DNA Bases and PNA Base Monomers to Gold Nanoparticles. J. Am. Chem. Soc. 2004, 126 (41), 13186−13187. (10) Henzl, M. T.; Agah, S. Divalent ion-binding properties of two avian β-parvalbumins. Proteins: Struct., Funct., Genet. 2006, 62 (1), 270−278. (11) Santos, H. A.; Manzanares, J. A.; Murtomaki, L.; Kontturi, K. Thermodynamic analysis of binding between drugs and glycosaminoglycans by isothermal titration calorimetry and fluorescence spectroscopy. Eur. J. Pharm. Sci. 2007, 32 (2), 105−114. (12) Frazier, R. A.; Papadopoulou, A.; Mueller-Harvey, I.; Kissoon, D.; Green, R. J. Probing Protein-Tannin Interactions by Isothermal

CONCLUSION

The seemingly straightforward binding process of [M2+ + EDTA] is very complex and involves many scientific concepts from freshman chemistry to upper-level courses: students review and strengthen their understanding of buffers as well as the relationships between pKa, pH, and protonation; students must consider the displacement of a proton from the binding ligand, a situation that also occurs often in an enzymatic binding pocket, and students must consider the thermodynamics of the displaced proton sequestered by the buffer. Additionally, students gained hands-on experience with the ITC to better prepare them for subsequent ITC experiments encountered in the biochemistry laboratory course.22 Ideas and suggestions for further development of this experiment include using several different buffers to illustrate the dependence on buffer ionization, changing the pH of reaction to demonstrate enthalpy associated with proton displacement in the binding pocket, and changing the chelator (e.g., EGTA instead of EDTA) to exhibit different chelatormetal specificities. D




Titration Microcalorimetry. J. Agric. Food Chem. 2003, 51 (18), 5189− 5195. (13) Arouri, A.; Garidel, P.; Kliche, W.; Blume, A. Hydrophobic interactions are the driving force for the binding of peptide mimotopes and Staphylococcal protein A to recombinant human IgG1. Eur. Biophys. J. 2007, 36 (6), 647−660. (14) Brockhaus, M.; Ganz, P.; Huber, W.; Bohrmann, B.; Loetscher, H.-R.; Seelig, J. Thermodynamic Studies on the Interaction of Antibodies with β-Amyloid Peptide. J. Phys. Chem. B 2007, 111 (5), 1238−1243. (15) Hofelich, T.; Wadsö, L.; Smith, A. L.; Shirazi, H.; Mulligan, S. R. The isothermal heat conduction calorimeter: A versatile instrument for studying processes in Physics, Chemistry, and Biology. J. Chem. Educ. 2001, 78 (8), 1080−1086. (16) Wadsö, L.; Li, X. A simple rate law experiment using a custombilt isothermal heat conduction calorimeter. J. Chem. Educ. 2008, 85 (1), 112−116. (17) Wadsö, L.; Li, Y.; Li, X. Isothermal titration calorimetry in the student laboratory. J. Chem. Educ. 2011, 88 (1), 101−105. (18) Isothermal Titration Calorimetry. http://en.wikipedia.org/wiki/ Isothermal_titration_calorimetry (accessed March 21, 2012). (19) Microcal Isothermal Titration CalorimetryMeasurement Principle. http://microcal.com/technology/itc.asp (accessed March 21, 2012). (20) Our ProductsNano ITC. http://www.tainstruments.com/ main.aspx?id=263&n=1&siteid=11 (accessed on March 21, 2012). (21) TA InstrumentsNano ITC for Biomolecular Binding. http:// www.youtube.com/tainstruments?v=cYj5IOELaVI&lr=1 (accessed March 21, 2012). (22) Wei, C.-C; Jensen, D.; Boyle, T.; O’Brien, L. C.; De Meo, C.; Shabestary, N.; Eder, D. J. Isothermal Titration Calorimetry and Macromolecular Modeling for the Interaction of Lysozyme and its Inhibitors. J. Chem. Educ., in press. DOI: 10.1021/ed5002569. (23) Quinn, C. F. Analyzing ITC Data for the Enthalpy of Binding Metal Ions to Ligands; TA Instruments: Lindon, UT, 2010. http:// www.tainstruments.com/pdf/MCAPN-201002%20ITC%20Metal%20Ions%20Binding%20to%20Ligands.pdf( accessed on March 21, 2012). (24) NanoAnalyze Software, version 2.2.0; TA Instruments: Lindon, UT, 2012. (25) Tellinghuisen, J. Designing isothermal titration calorimetry experiments for the study of 1:1 binding: Problems with the “standard protocol. Anal. Biochem. 2012, 424 (2), 211−220. (26) Roig, T.; Backman, P.; Olofsson, G.; et al. Ionization enthalpies of some common zwitterionic hydrogen-ion buffers (HEPES, PIPES, HEPPS, and BES) for biological research. Acta Chem. Scand. 1993, 47 (9), 899−901.

E


M2+â¢EDTA Binding Affinities: A Modern Experiment in

Recommend Documents

M2+â¢EDTA Binding Affinities: A Modern Experiment in