Communication Cite This: J. Chem. Educ. XXXX, XXX, XXX-XXX
pubs.acs.org/jchemeduc
Experimental Determination of pKa Values and Metal Binding for Biomolecular Compounds Using 31P NMR Spectroscopy Mason A. Swartz, Philip J. Tubergen, Chad D. Tatko, and Rachael A. Baker* Department of Chemistry and Biochemistry, Calvin College, Grand Rapids, Michigan 49506, United States S Supporting Information *
ABSTRACT: This lab experiment uses 31P NMR spectroscopy of biomolecules to determine pKa values and the binding energies of metal/biomolecule complexes. Solutions of adenosine nucleotides are prepared, and a series of 31P NMR spectra are collected as a function of pH and in the absence and presence of magnesium or calcium ions. The chemical shifts in the spectrum change depending on the protonation state of the phosphate groups and the nucleotide/metal interaction. Because 31P NMR spectroscopy is used, the resulting data are clean and readily accessible for student analysis. The use of 31P NMR spectroscopy presents a rich opportunity to deepen student engagement with NMR spectroscopy and spectral analysis. Furthermore, the inclusion of biomolecules and metal binding exposes students to important and underrepresented concepts that bridge organic chemistry and biochemistry. Students will learn how to prepare, collect, and analyze nonhydrogen samples using NMR spectroscopy, identify an unknown pKa, and calculate binding energies for a metal complex. This experiment is suitable for students in a second-year undergraduate organic chemistry course and could also be used in an analytical chemistry, bioorganic chemistry, or an advanced instrumentations laboratory course. KEYWORDS: Second-Year Undergraduate, Upper-Division Undergraduate, Analytical Chemistry, Organic Chemistry, Hands-On Learning/Manipulatives, Bioorganic Chemistry, NMR Spectroscopy, Nucleic Acids/DNA/RNA, pH, Quantitative Analysis
T
substrates for a myriad of metabolic and enzymatic reactions in vivo.3−5 Notably, complexes are formed between metal ions and adenine nucleotides that are essential for their functioning, particularly in enzymatic reactions.6−9 Two of the most biologically relevant metals for these processes are magnesium and calcium.10 There are multiple equilibrium relationships between the nucleotide and cation, as shown for ATP in Figure 1. However, while the equilibrium relationships between the metals and various adenine nucleotides are quite complex, many of the nuances of nucleotide/metal binding are not observed by 31P NMR spectroscopy or lie outside of the pH range investigated in this lab experiment.11−13 The simplicity of the system under the conditions described in this lab experiment allows students to effectively engage these important concepts at a second-year undergraduate level. Titrations of adenine nucleotides are executed while following only the 31P NMR signals. The use of 31P NMR spectroscopy significantly decreases associated costs because the data can be collected in water with a minimum of deuterated solvents. D2O, necessary to obtain a lock signal, is typically present at only 10% total volume. Furthermore,31P NMR spectroscopy produces clean and easy-to-analyze data. The protonation and deprotonation equilibrium described in
he following lab experiment is a reimagining of the standard titration lab experiment using NMR spectroscopy, biomolecules, and extensive data processing. NMR spectroscopy is a robust and versatile tool for analyzing chemical compounds. Furthermore, the use of NMR spectroscopy in biochemical investigations represents an area of exponential growth and potential, particularly because of its use in querying structural and dynamic events. While the possibilities of NMR spectroscopy applications to biochemistry are evident, bridging the use of NMR spectroscopy from small organic molecules to biochemically relevant molecules in the educational framework represents a significant challenge. The primary locus of NMR spectroscopy is traditionally housed in the organic sequence. This updated lab experiment uses a classic organic chemistry lab experiment to highlight the relevance of NMR spectroscopy in bioorganic chemistry. The lab experiment described herein advances previous laboratories that calculate pKa values by introducing heteronuclear NMR data collection, utilization of biomolecules, and substantial data analysis.1 The lab also engages multiple equilibria thermodynamic calculations absent from a previous lab approach that only examined metal binding via chemical shift.2 The biomolecules chosen for this lab experiment are adenine nucleotides, including adenosine 5′-triphosphate (ATP), adenosine 5′-diphosphate (ADP), and adenosine 5′-monophosphate (AMP). Adenine nucleotides are widely involved in energy transfer and responses to various cellular stimuli and are © XXXX American Chemical Society and Division of Chemical Education, Inc.
Received: July 12, 2017 Revised: September 15, 2017
A
DOI: 10.1021/acs.jchemed.7b00508 J. Chem. Educ. XXXX, XXX, XXX−XXX
Journal of Chemical Education
Communication
Figure 1. Multiple equilibrium relationships for ATP in the presence of magnesium. Nomenclature for various protonated and metal binding states as well as the equilibrium between them are given in the figure. This same nomenclature is used throughout for equations and calculations.
■
Figure 1 will impact the chemical shift of the α, β, and γ phosphates of adenine nucleotides in a pH-dependent manner. Therefore, the peak shifts observed by 31P NMR spectroscopy can be fit to determine a pKa using eq 1, which is shown for ATP. For more information, see the full equation derivation in the Supporting Information. δobs =
EXPERIMENTAL SECTION Before coming to lab, students were given background information that provided context for complex equilibria in biology and the fundamentals of 31P NMR spectroscopy. The students then answered a series of prelab questions that were carefully designed to draw their attention to similarities and differences between 31P NMR spectroscopy and other common types of NMR spectroscopy. The questions also helped the instructor assess preconceived notions students had about the system they would be studying and the data to be analyzed. For details, see the Student Handout provided in the Supporting Information. At the start of the lab experiment, students were grouped into pairs and assigned a nucleotide or nucleotide/cation combination to study. A one-dimensional 31P NMR scan takes approximately the same time as a typical 1H NMR experiment. As a result, three groups could be reasonably accommodated in a 3−4 h lab period. The measurements were done in water with 10% D2O incorporated to obtain a lock. An internal reference standard of 85% phosphoric acid in a separate capillary was used for each measurement. After checking the initial pH of the system, students adjusted the pH to 3, and then NaOH was used to raise the pH slowly to 10 or 11. NMR spectra were collected after every 0.5 pH units except near the pKa. Additional details as well as a complete data set are provided in the Instructor’s Notes in the Supporting Information. Chemical shifts were assigned by students on the basis of the table provided in the Student Handout (Supporting Information). Students analyzed and plotted their data to determine the pKa of their system. Finally, student data were pooled for binding energy calculations. A series of postlab questions, which emphasized the connection between organic chemistry and NMR spectroscopy of biological systems, were used to assess student understanding of the lab experiment and its applications for living systems.
(δ ATP4−)(10(pH − pKa)) + δ(ATP)H3− 1 + 10(pH − pKa)
(1)
In the presence of a cation, the binding of the cation (magnesium in Figure 1) can alter the acid−base equilibrium of the terminal phosphate of the adenine nucleotide.14 This combination of equilibrium gives a meaningful outcome. The determined change in the pKa of the nucleotide reflects the difference in association of the cation with the conjugate acid and base species, as illustrated for magnesium and ATP in eq 2. Mg H − = pK H pKMg:(ATP)H + log KMg:(ATP)H − (ATP)H3 − Mg − log KMg:ATP 2−
(2)
The change in pKa for the system in the absence and presence of the cation can be used to quantify the energy difference related to the strength of cation binding using eq 3. H H ΔΔG = 2.303 × RT × (pK(ATP)H 3 − − pKMg:(ATP)H−)
(3)
These simple mathematical analyses facilitate the achievement of rich pedagogic goals, including that students will be able to (1) collect and analyze heteroatom data using 31P NMR spectroscopy, (2) understand chemical shift changes and their usefulness for determining the pKa of a system, (3) identify the pKa for a biomolecule and the binding energy for a metal/ biomolecule complex, and (4) describe the usefulness of organic techniques for probing biological systems. Ultimately, through exploring chemical shifts using NMR spectroscopy, students deepen their understanding of thermodynamic equilibrium in a biological context that is suitable for secondyear organic chemistry or upper-division bioorganic or biochemistry students.
■
HAZARDS
As with many laboratory procedures, personal protective equipment is required, specifically gloves and safety glasses. Both adenosine 5′-triphosphate and CaCl2 are skin irritants; CaCl2 is a serious eye irritant. The remaining reagents, D2O, B
DOI: 10.1021/acs.jchemed.7b00508 J. Chem. Educ. XXXX, XXX, XXX−XXX
Journal of Chemical Education
Communication
Figure 2. Stacked series of 31P NMR spectra for the titration of ATP showing the chemical shift of the α, β, and γ phosphates. The reference standard (85% phosphoric acid) is assigned a chemical shift of 0.0 ppm. The representative student data are shown from low pH (bottom) to high pH (top). The data for this particular experiment were collected at pH 2.14, 2.93, 3.98, 4.45, 5.16, 6.06, 7.22, 7.86, 8.28, 9.16, 9.81, 10.53, 11.23, 12.00, 12.64, and 12.79.
adenosine 5′-diphosphate, adenosine 5′-monophosphate, and MgCl2, do not pose significant hazards. The acids (phosphoric acid and HCl) and base (NaOH) used in the titration pose a corrosive and caustic risk, requiring personal protective equipment.
■
RESULTS AND DISCUSSION The complete data set collected by students, including three nucleotides and two divalent binding partners, is rich for analysis. For illustration, Figure 2 shows data collected by students during the pH titration of ATP. As expected, multiplicity was observed for the α, β, and γ phosphates. Under certain conditions, the presence of the cation may cause exchange-induced line broadening, but this did not inhibit chemical shift data collection or analysis.15 Using the reference peak, the three phosphate shifts were readily assigned and could be fit to determine the amount of protonated and deprotonated species present. Once the chemical shifts were recorded as a function of pH, the data were fit to determine the pKa of the system using eq 1. An example of chemical shift fitting is given for the γ phosphate from ATP in Figure 3. The complete data set for calculated pKa values is provided in Table 1. The experimentally determined values by students were close to the literature reported values; small changes arose from temperature and concentration discrepancies.12,16,17 Using the pKa of each system, students determined the difference in cation binding to the protonated and deprotonated forms of the nucleotide. While stronger interactions occur with the fully deprotonated species, there will be some interaction between the cation and nucleotide in the monoprotonated form (Figure 1). When students fit the data in the presence of a metal ion, a shift was observed in the
Figure 3. Chemical shift of the γ-ATP resonance over the course of a titration. Data are shown for three separate student determinations of the pKa. The data are represented in orange, green, or blue for the individual students, and the line reflects the curve fit to the data. The x error represents the error from measuring the pH on the pH meter. The y error is the 95% confidence interval from the curve fitting. The average and standard error for the pKa values determined from these three particular experiments were 6.58 ± 0.13.
calculated pKa, which delineated this preference. For example, there is an increase in binding energy of 11.7 kJ mol−1 between the conjugate base and acid form of ATP. This lab experiment has been run with 20 students over multiple years. After completing the lab experiment, all students were able to answer the postlab questions at a developing or accomplished level as described by the grading rubric (Supporting Information). The postlab questions test student understanding of 31P NMR spectroscopy as well as their ability to think about their results in a biologically relevant context. C
DOI: 10.1021/acs.jchemed.7b00508 J. Chem. Educ. XXXX, XXX, XXX−XXX
Journal of Chemical Education
Communication
Table 1. pKa Values Determined from Individual Phosphorous Resonances Alone (SE)a
Magnesium Present (SE)a
Calcium Present (SE)a
Nucleotide
α
β
γ
α
β
γ
α
β
γ
ATP ADP AMP
6.80 (0.20) 6.41 (0.03) 6.33 (0.02)
6.60 (0.09) 6.47 (0.01) b
6.66 ± 0.03 b b
4.70 (0.10) 5.32 (0.02) 5.81 (0.02)
5.10 (0.10) 5.32 (0.02) b
4.58 (0.03) b b
6.40 (0.10) 5.57 (0.06) 6.06 (0.03)
6.05 (0.07) 5.78 (0.05) b
6.13 (0.06) b b
a
Representative student data are shown along with the standard error. bResonance not present for this particular nucleotide. (7) Sprang, S. R.; Coleman, D. E. Invasion of the Nucleotide Snatchers. Cell 1998, 95 (2), 155−158. (8) Zhang, B.; Zhang, Y.; Wang, Z.; Zheng, Y. The Role of Mg2+ Cofactor in the Guanine Nucleotide Exchange and GTP Hydrolysis Reactions of Rho Family GTP-Binding Proteins. J. Biol. Chem. 2000, 275 (33), 25299−25307. (9) Dittrich, M.; Hayashi, S.; Schulten, K. On the Mechanism of ATP Hydrolysis in F1-ATPase. Biophys. J. 2003, 85 (4), 2253−2266. (10) Cox, J. R.; Ramsay, O. B. Mechanisms of Nucleophilic Substitution in Phosphate Esters. Chem. Rev. 1964, 64 (4), 317−352. (11) Corfu, N. A.; Sigel, H. Acid-Base Properties of Nucleosides and Nucleotides as a Function of Concentration. Eur. J. Biochem. 1991, 199 (3), 659−669. (12) Sigel, H.; Griesser, R. Nucleoside 5-Triphosphates: SelfAssociation, Acid-Base, and Metal Ion-Binding Properties in Solution. Chem. Soc. Rev. 2005, 34 (10), 875−900. (13) Jiang, L.; Mao, X.-A. NMR Evidence for Mg(II) Binding to N1 of ATP. Spectrochim. Acta, Part A 2001, 57 (8), 1711−1716. (14) Cohn, M.; Hughes, T. R. Nuclear Magnetic Resonance Spectra of Adenosine Di- and Triphosphate: II. Effect of Complexing with Divalent Metal Ions. J. Biol. Chem. 1962, 237 (1), 176−181. (15) Szabó, Z. Multinuclear NMR Studies of the Interaction of Metal Ions with Adenine-Nucleotides. Coord. Chem. Rev. 2008, 252 (21−22), 2362−2380. (16) Pecoraro, V. L.; Hermes, J. D.; Cleland, W. W. Stability Constants of Magnesium and Cadmium Complexes of Adenine Nucleotides and Thionucleotides and Rate Constants for Formation and Dissociation of Magnesium-ATP and Magnesium-ADP. Biochemistry 1984, 23 (22), 5262−5271. (17) Jaffe, E. K.; Cohn, M. 31P Nuclear Magnetic Resonance Spectra of the Thiophosphate Analogs of Adenine Nucleotides; Effects of pH and Mg2+ Binding. Biochemistry 1978, 17 (4), 652−657.
From grading prelab and postlab questions, it was evident that students had an initial perception that 31P NMR spectroscopy would be slow and lack multiplicity, similar to 13C NMR spectroscopy. However, they were able to apply previous knowledge from exposure to 1H and 13C NMR spectroscopy to collect, assign, and analyze the spectral data. Students reported enjoying the lab experiment, particularly because of the biological relevance and the clean data, which allowed them to focus on the key features of the spectrum and the required analysis.
■
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available on the ACS Publications website at DOI: 10.1021/acs.jchemed.7b00508. Derivation of pKa formula (PDF, DOCX) Instructor notes (PDF, DOCX) Student handout (PDF, DOCX) Spreadsheet for data analysis and curve fitting (XLSX)
■
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Rachael A. Baker: 0000-0002-3803-1828 Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS C.D.T. and R.A.B. thank the Calvin College Alumni Association and the National Science Foundation (CHE 0922973, CHE 963433) for supporting this research. P.J.T. and M.A.S. thank Calvin College for research fellowships.
■
REFERENCES
(1) Gift, A. D.; Stewart, S. M.; Kwete Bokashanga, P. Experimental Determination of pKa Values by Use of NMR Chemical Shifts, Revisited. J. Chem. Educ. 2012, 89 (11), 1458−1460. (2) Hurst, M. O.; Orvis, J. A. Effect of Magnesium Chelation on the 31 P NMR Spectra of ATP: An Undergraduate Biochemistry Laboratory Experiment. Chem. Educ. 2005, 10 (5), 359−362. (3) Westheimer, F. H. Monomeric Metaphosphates. Chem. Rev. 1981, 81 (4), 313−326. (4) Corriden, R.; Insel, P. A. Basal Release of ATP: An AutocrineParacrine Mechanism for Cell Regulation. Sci. Signaling 2010, 3 (104), re1. (5) Lazarowski, E. R.; Sesma, J. I.; Seminario-Vidal, L.; Kreda, S. M. Molecular Mechanisms of Purine and Pyrimidine Nucleotide Release. Adv. Pharmacol. 2011, 61, 221−261. (6) Anastassopoulou, J.; Theophanides, T. The Role of Metal Ions in Biological Systems and Medicine. In Bioinorganic Chemistry: An Inorganic Perspective of Life; Kessissoglou, D. P., Ed.; Springer Netherlands: Dordrecht, 1995; pp 209−218. D
DOI: 10.1021/acs.jchemed.7b00508 J. Chem. Educ. XXXX, XXX, XXX−XXX
