How Do Structure and Charge Affect Metal ... - ACS Publications

LABORATORY EXPERIMENT pubs.acs.org/jchemeduc

How Do Structure and Charge Affect Metal-Complex Binding to DNA? An Upper-Division Integrated Laboratory Project Using Cyclic Voltammetry Agnieszka Kulczynska, Reed Johnson, Tony Frost, and Lawrence D. Margerum* Department of Chemistry, University of San Francisco, San Francisco, California 94117, United States

bS Supporting Information ABSTRACT: An advanced undergraduate laboratory project is described that integrates inorganic, analytical, physical, and biochemical techniques to reveal differences in binding between cationic metal complexes and anionic DNA (herring testes). Students were guided to formulate testable hypotheses based on the title question and a list of different metal complexes. Student teams synthesized the target complexes, such as tris(1,10phenanthroline)cobalt(III) or tris(2,20 -bipyrydyl)cobalt(III), and characterized them by voltammetry and spectroscopy. Separately, DNA stock solutions were prepared and analyzed via published spectroscopic methods. Aliquots of the DNA solutions, added into a metal-complex solution, gave decreases in the cyclic voltammetry peak currents due to the slower diffusion rate of the DNAmetal complex. A nonlinear curve fit analysis of the 1:1 binding isotherms confirmed the literature result (unknown to students) of larger binding constants for the phenanthroline complex due to intercalative binding. Student team results were shared in a group meeting and assessment was by group reports and individual portfolios. The project was an effective way to link the various laboratory techniques common to the chemical disciplines and encouraged team building in a research atmosphere. KEYWORDS: Upper-Division Undergraduate, Interdisciplinary/Multidisciplinary, Laboratory Instruction, Hands-On Learning/ Manipulatives, Inquiry-Based/Discovery Learning, Coordination Compounds, Electrochemistry, Nucleic Acids/DNA/RNA, Oxidation/Reduction, Spectroscopy used as therapeutic agents.4 The application of voltammetry methods for quantitative binding of metal chelates to DNA provides a useful complement to more time-consuming techniques, such as equilibrium dialysis.5,6 In general, the electrochemical methods are dependent upon the diffusion rate of the metal complex. An electrochemically active metal-complex binding to the slower moving DNA macromolecule will have a smaller diffusion current compared to the free complex. The magnitude of the drop in current should depend upon both electrostatic and nonelectrostatic contributions to the equilibrium binding constant, so it will be dependent upon the structure and charge of the metal complex.5,7,8 This becomes the focal point of the integrated lab project here as we adapt procedures from the Thorp group on the use of voltammetric data for DNA binding8 and draw upon the authors’ experience with metal-complex binding to polyamine dendrimer macromolecules.9,10 This student laboratory project starts with a question: “How do structure and charge affect metal-complex binding to DNA?” The instructor provides a list of metal complexes that have the same charge and different ligands or are structurally similar metal

M

ost chemistry majors at our liberal arts university complete an upper-level capstone course that consists of lectures in instrumental analysis, plus laboratory experiments focusing on one instrument at a time. We look to develop more projectbased, integrated experiments that reflect real-world research on a variety of instruments. The purpose of the project here is to combine some traditional laboratory topics in physical chemistry (electrochemistry, equilibrium binding), inorganic chemistry (metal-complex synthesis and characterization), and biochemistry (spectroscopic characterization of DNA solutions) into one package to impress upon students the links between chemical disciplines. The project design draws heavily upon literature results and methods, yet links techniques together and extends others, which may appeal to instructors seeking an integrated approach for upper-level laboratory courses with a biochemical flavor. Parts of this project are modeled after guided-inquiry methods designed to stimulate and engage students in cooperative learning groups.1 The binding of small molecules or ions to nucleic acids is of central concern to a broad cross section of researchers and finds its way into the undergraduate biophysical laboratory curriculum.2,3 Negatively charged biomolecules offer excellent ligands for binding of metal ions and for selected metal complexes that are Copyright r 2011 American Chemical Society and Division of Chemical Education, Inc.

Published: April 18, 2011 801

dx.doi.org/10.1021/ed100652s | J. Chem. Educ. 2011, 88, 801–805

Journal of Chemical Education complexes that differ in overall charge. The class picks a testable hypothesis as to why one metal complex may bind more strongly than the other to polyanionic DNA. Smaller student teams each synthesize one metal complex and characterize the redox chemistry using cyclic voltammetry (CV). Meanwhile, each team also prepares a stock solution of DNA and analyzes it using standard UV methods. Finally, students carry out manual titrations and record the drop in CV diffusion peak currents with added DNA. The best data sets are transferred to a curve-fitting software program to produce an estimate of the 1:1 binding constants between the metal complex and DNA. At the end, groups come together to present data, discuss results, and produce a written report. Students gain experience in project work, inorganic synthesis, quantitative voltammetry, DNA manipulations, nonlinear curve-fitting techniques, and group “research” presentations.

’ EXPERIMENTAL SECTION Reagents

All reagents were ACS grade or above. Methanol (g99%); cobalt(II)chloride hexahydrate, CoCl2 3 6H2O (98.0102.0%); 2,20 -bipyridine (g99%); 1,10-phenanthroline hydrate (g99%); liquid bromine (reagent grade); perchloric acid, HClO4 (70%); 2-amino-2-(hydroxymethyl)-1,3-propanediol hydrochloride (Trizma or Tris, g99.9%); sodium chloride (g99.5%); and DNA (sodium salt Type XIV from herring testes) were used as received from Sigma-Aldrich. Millipore Milli-Q water (18.2 mΩ cm) was used to prepare all aqueous solutions. All buffer solutions were 50 mM NaCl/5 mM Tris/pH 7 (adjusted with HCl). The tris(1,10-phenanthroline)cobalt(III) and tris(2,20 bipyridyl)cobalt(III) perchlorate trihydrate complexes were prepared according to literature procedure.11 Bromine water was prepared by pouring the vapors from a bromine reagent bottle into a 100 mL bottle containing 50 mL of water, followed by mixing.

’ PROCEDURE AND INSTRUMENTATION To avoid dilution effects during titrations, salts of DNA and metal complexes were weighed and transferred into a volumetric flask and filled with buffer solution to give 6 mM DNA (defined as the concentration of nucleotide phosphates, NP) and 0.1 mM metal complex.8 After standing for 20 min, the stock solution was sonicated for 1 min increments. Students were required to find a standard method for a nucleic acid UV analysis using the HP 8350 UVvis diode array, such as the primary sources found within Wikipedia.12 The electrochemical cells, electrodes, and polishing kits were purchased from BAS (West Lafayette, IN). Electrochemical measurements and titrations were performed in a one-compartment cell using a glassy carbon (GC) working electrode (polished according to the supplier), an auxiliary electrode (Pt wire), and a RE-5 Ag/AgCl reference electrode. The solution was deoxygenated with water-saturated argon gas for ca. 5 min. Cyclic voltammetry (CV) was carried out using a Princeton Applied Research (PAR) model 283 potentiostat controlled by PowerCV software. The CV theory and practice has been described in this Journal,1315 along with an affordable instrument option.16 GraphPad Prism 4 software was used for the nonlinear regression analysis and graphing of the binding isotherms.

LABORATORY EXPERIMENT

Figure 1. Two metal complexes with the same charge.

’ HAZARDS Concentrated perchloric acid is highly corrosive and oxidizing. Pure liquid bromine is highly toxic by inhalation, ingestion, or skin contact. It causes severe burn and is a lachrymator. Methanol is extremely flammable and toxic by inhalation. Methanol may be fatal or cause blindness if swallowed. Cobalt(II)chloride hexahydrate, 2,20 -bipyridine, 1,10-phenanthroline hydrate, and Tris may cause irritation to skin, eyes, and respiratory tract and may be harmful if swallowed or inhaled. Use rubber or nitrile gloves in a fume hood. We have not encountered any problems; yet, perchlorate salts of metal complexes or ions are potentially shock sensitive. One should only isolate small amounts and they should not be dried or stored under vacuum. ’ RESULTS AND DISCUSSION A typical student hypothesis for this project is that metal complexes with the same charge, for example, tris(1,10-phenanthroline)cobalt(III) [Co(phen)3]3þ and tris(2,20 -bipyrydyl)cobalt(III) [Co(bpy)3]3þ (Figure1), will give the same binding to anionic DNA. Scans labeled “a” in Figure 2 are student-generated diffusion current versus potential (i vs E) curves for the CV experiment on solutions of [Co(bpy)3]3þ and [Co(phen)3]3þ in the absence of DNA. The working electrode potential program starts at 0.4 V and proceeds to more negative, reducing, potentials at a constant scanning rate. The result is a peak in the diffusion current as the Co(III) complex is reduced to Co(II) near the electrode surface. Reversing the potential at 0.2 V and returning to the starting point results in an anodic peak current as the Co(III) complex reforms. Measurements taken from these curves give three important parameters: (i) the standard reduction or half-wave potential from the peaks, E1/2 = 0.5(Ep,cathodic þ Ep,anodic), (ii) the difference in peak potentials (ΔE = 57 mV for a reversible one-electron system), and (iii) the ratio of cathodic peak to anodic peak currents ipc/ipa (equal to unity for a reversible system). Typical student results in the absence of DNA (at 50 mV s1 scan rates) are ΔE = 0.110 V (vs Ag/AgCl), ΔE = 67 mV, ipc/ipa = 0.99 for [Co(bpy)3]3þ and E1/2 = 0.166 V, ΔE = 68 mV, ipc/ipa = 0.98 for [Co(phen)3]3þ, which are consistent with reversible one-electron redox couples.7 Students discover the linear region of the RandleSelvich equation at 25 °C for cyclic voltammetry, ip = (2.69 105)n3/2Av1/2CD1/2, by plotting ip (peak current) versus scan rate ν1/2, where n is the number of electrons transferred, A is the electrode surface area, C is concentration, and D is the diffusion coefficient. Most plots are linear for scan rates 5200 mV s1, but fall off at higher rates (data not shown). The linear region is therefore under diffusion control. 802

dx.doi.org/10.1021/ed100652s |J. Chem. Educ. 2011, 88, 801–805

Journal of Chemical Education


Figure 2. Cyclic voltammograms at 50 mV s1 of (A) 0.1 mM [Co(bpy)3]3þ and (B) 0.1 mM [Co(phen)3]3þ in 50 mM NaCl and 5 mM Tris adjusted to pH 7 at GC electrodes: curve a is the absence of DNA; curve b is after the first addition of DNA stock solution; and curve c is after the final addition of similar aliquots of DNA stock solution.

Table 1. Nonlinear Curve-Fitting Output to eq 1 from GraphPad Prism 4 Parameter Best-Fit Valuesa

[Co(bpy)3]3þ

[Co(phen)3]3þ

K = 406.4

K = 751.1

N = 0.55

N = 1.74

Standard Error

K = 24.0 N = 0.02

K = 76.1 N = 0.07

95% Confidence

K = 355.2 to 457.5

K = 588.9 to 913.3

Interval Goodness of Fit

Figure 3. Data points and curve fits to eq 1 (solid line) of the difference between CV peak currents for bound (b) and unbound (0) metal complexes as a function of added herring testes DNA in 50 mM NaCl and 5 mM Tris at pH 7. NP is concentration in terms of nucleotide phosphates.

K½DNA 1 þ K½DNA

N = 1.88 to 1.59 Degrees of Freedom =15

R2 = 0.9971

R2 = 0.9865

a

K is the 1:1 binding constant between metal complex and nucleotide phosphates and N = (ib2 i02) in eq 1.

Scheme 1. Difference in Formal Potential of the Fully Bound Probe (Ebo0 ) and the Free Probe

Next, students add DNA stock solution via calibrated digital pipet and mix before obtaining scans “b” through “c” in Figure 2 (not all scans shown). The decrease in the peak current is direct evidence for metal-complex binding to the slower diffusing DNA because there is no change in metal-complex concentration under the titration conditions (see the Supporting Information). A recent electrochemical experiment described how the diffusion coefficient of ferrocenes changed as a function of binding to micelles of different charge and structure.17 Thorp and co-workers have discussed the limitations of the CV technique for determination of diffusion coefficients of macromolecules.18 One may also use differential-pulse voltammetry (DPV) to monitor diffusion currents and this gives similar results. At this point the equations for using CV data to obtain small molecule conditional binding constants (K) to macromolecules8,18 is introduced to the students: i2 i20 ¼ ði2b i20 Þ

N = 0.58 to 0.51 Degrees of Freedom =15

software program to find fitted values to the nonlinear data for the two variables, K and the saturation binding current ib (this value is not experimentally accessible for small values of K, so ib2 i02 is the other variable). Typically, groups find marginal fits to the first data set as they did not collect enough data points or were not precise with small volume digital pipets. A series of guiding questions or informal group discussion leads them to revise the experimental design to obtain better data. A simultaneous “global fit” of two or more subsequent data sets often leads to more reliable multiparameter fits.19 Student results for the titrations are presented in Figure 3 and Table 1. The binding

ð1Þ

The binding isotherm in eq 1 can be visualized by plotting the difference between peak currents measured after and before each addition of DNA, i2 i02 versus [DNA] (see the Supporting Information for the entire derivation). Students enter eq 1 into a 803



Figure 4. Dependence of CV half wave potential on the molar ratio R of [DNA]:[CoL3]3þ for metal complexes in pH 7 Tris buffer (5 mM) with added NaCl (50 mM). The solid lines are added for clarity.

constants here are somewhat lower than found in previous work using calf thymus DNA, although the ratios are similar.8 The herring testes DNA is less costly than calf thymus DNA and is used for electrochemical studies of metal-complex binding to DNA at microelectrodes.6 Several students point out that the peak potentials of some metal complexes shift during the titration (such as for [Co(phen)3]3þ in Figure 2). If purely electrostatic binding dominates, then 3þ complexes should bind more strongly than the electrochemically produced 2þ complexes. The difference in formal potential of the fully bound probe (Ebo0 ) and the free probe (Efo0 ) is related to the ratio of binding constants through the Nernst equation as shown in Scheme 1 (modified from refs 7, 20). Figure 4 depicts the half-wave potentials for the metal complexes as a function of the mole ratio R (DNA:metal complex). The half-wave potential at the end of the titration is taken as Ebo0 , whereas the initial value is the free probe potential Efo0 . Surprisingly, with herring testes DNA, the half-wave potentials for [Co(bpy)3]3þ/2þ show almost no shift (5 ( 3 mV, estimated error) with excess DNA, meaning that K2þ/K3þ is unity under these conditions. This result is different from the literature values for [Co(bpy)3]3þ/2þ titrations with calf thymus DNA that gave a large negative shift for Ebo0 Efo0 and a K2þ/K3þ ratio of 0.6, meaning that the 3þ ion was bound 1.7 times more strongly than the 2þ ion, consistent with electrostatic theory. The other probe, [Co(phen)3]3þ/2þ, shows positive shifts of 20 mv, giving K2þ/K3þ equal to 2.18, meaning the 2þ ion binds more strongly than the 3þ ion. This result is similar to calf thymus DNA using the same probe, K2þ/K3þ = 1.94.7 In summary, student groups clearly see that their initial hypothesis is not supported by the data. Barton and co-workers explained that one phen ligand on a metal complex may bind between adjacent DNA base pairs (intercalation effect) with the remaining ligands disposed along the more hydrophobic major groove compared to the charged sugarphosphate backbone.21 These arguments favor 2þ over 3þ ion binding of tris-chelated metal phen complexes to DNA as seen here. At present, we cannot explain why calf thymus and herring testes DNA give different K2þ/K3þ ratios for [Co(bpy)3]3þ/2þ (for example, the guaninecytosine content, that may affect nonelectrostatic binding, are similar).22 This suggests the need for further study of metal chelate binding to DNA from different sources.


’ CONCLUSIONS AND EXTENSIONS This project-based lab gives students and instructors many pathways to test the structureactivity question posed at the beginning. We find student groups struggle with almost all aspects of the experimental design and implementation. In part this may reflect the lack of such practice in our curriculum, plus unrealistic student expectations in a lab course. Thus, one important outcome is to schedule enough exploration time for student groups to be coached on how to formulate a hypothesis, prepare and analyze DNA solutions, master the CV technique, and improve upon the DNA titrations. Replication is an idea that student groups usually come up with knowing they have enough time.23 We also coach and assess group presentations, group reports, and individual portfolios for the project. Several extensions may appeal to instructors who have more students or who want to emphasize certain parts of the project. First, other metal complexes such as [Fe(bpy)3]2þ, [Fe(phen)3]2þ, [Ru(NH3)6]3þ and the ferrocenes have been used as electrochemical probes for nucleic acids.6,24,25 One could also test the effect of ionic strength on binding constants. Lowering the ionic strength increases the binding constants as predicted by electrostatic theory.8,26 Finally, one is not limited to DNA as the macromolecule of interest. Voltammetric methods were used to monitor Cu(II) Schiff base complexes or Cu(II) ions binding to bovine serum albumin (BSA).3,27 If the focus is on polymer or nanochemistry, one could choose a variety of anionic or cationic macromolecules for binding studies by voltammetric methods, such as the polystyrene sulfonates2830 or polyamine dendrimers whose diffusion and electrostatic binding to anionic metal complexes change with size.9 ’ ASSOCIATED CONTENT

bS

Supporting Information Student handout and notes for the instructor. This material is available via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected].

’ ACKNOWLEDGMENT We thank USF students in CHEM 410 for testing this project. Support from the NSF for electrochemical equipment (CHE0216617) and the USF Faculty Development Fund for undergraduate research (R.J.) are gratefully acknowledged. ’ REFERENCES (1) Moog, R. S.; Spencer, J. N., POGIL: Process Oriented Guided Inquiry Learning, ACS Symposium Series 288. Oxford University Press: New York, 2008. (2) Matthew, A. F.; Danielle, J.; Daniel, A. R. J. Chem. Educ. 2002, 79, 374–376. (3) Jie, L. J. Chem. Educ. 2004, 81, 395–397. (4) Zhang, C. X.; Lippard, S. J. Curr. Opin. Chem. Biol. 2003, 7, 481–489. (5) Carter, M. T.; Bard, A. J. J. Am. Chem. Soc. 1987, 109, 7528–7530. (6) Aslanoglu, M.; Isaac, C. J.; Houlton, A.; Horrocks, B. R. Analyst 2000, 125, 1791–1798. 804




(7) Carter, M. T.; Rodriguez, M.; Bard, A. J. J. Am. Chem. Soc. 1989, 111, 8901–8911. (8) Welch, T. W.; Thorp, H. H. J. Phys. Chem. 1996, 100, 13829–13836. (9) Kulczynska, A.; Frost, T.; Margerum, L. D. Macromolecules 2006, 39, 7372–7377. (10) Frost, T.; Margerum, L. D. Macromolecules 2010, 43, 1218–1226. (11) Dollimore, L. S.; Gillard, R. D. J. Chem. Soc., Dalton Trans. 1973, 933–940. (12) Wikipedia Nucleic acids analysis. http://en.wikipedia.org/wiki/ Nucleic_acids_analysis (accessed Mar 2011). (13) Mabbott, G. A. J. Chem. Educ. 1983, 60, 697–702. (14) Kissinger, P. T.; Heineman, W. R. J. Chem. Educ. 1983, 60, 702–706. (15) Van Benschoten, J. J.; Lewis, J. Y.; Heineman, W. R.; Roston, D. A.; Kissinger, P. T. J. Chem. Educ. 1983, 60, 772–776. (16) Amend, J. R.; Stewart, G.; Kuntzleman, T. S.; Collins, M. J. J. Chem. Educ. 2009, :: 86, 1080–1081. € (17) Koca, A.; Uce, M.; Ozkaya, A. R.; S-ahin, M. Chem. Educ. 2007, 12, 185–189. (18) Welch, T. W.; Corbett, A. H.; Thorp, H. H. J. Phys. Chem. 1995, 99, 11757–11763. (19) Motulsky, H. J.; Christopoulos, A. Global nonlinear regression with Prism 4. http://graphpad.com/articles/P4Global.pdf (accessed Mar 2011). (20) Kaifer, A. E.; Bard, A. J. J. Phys. Chem. 1985, 89, 4876–4880. (21) Barton, J. K.; Goldberg, J. M.; Kumar, C. V.; Turro, N. J. J. Am. Chem. Soc. 1986, 108, 2081–2088. (22) Deng, H.; Bloomfield, V. A.; Benevides, J. M.; G., J. T., Jr. Nucleic Acids Res. 2000, 28, 3379–3385. (23) DeMeo, S. Chem. Educ. 2007, 12, 387–391. (24) Shah, A.; Zaheer, M.; Qureshi, R.; Akhter, Z.; Nazar, M. F. Spectrochim. Acta, Part A 2010, 75A, 1082–1087. (25) Shah, A.; Qureshi, R.; Janjua, N. K.; Haque, S.; Ahmad, S. Anal. Sci. 2008, 24, 1437–1441. (26) Kalsbeck, W. A.; Thorp, H. H. Inorg. Chem. 1994, 33, 3427–3429. (27) Boghaei, D. M.; Farvid, S. S.; Gharagozlou, M. Spectrochim. Acta, Part A 2007, 66A, 650–655. (28) Jiang, R.; Anson, F. C. J. Phys. Chem. 1991, 95, 5701–5706. (29) Jiang, R.; Anson, F. C. J. Phys. Chem. 1992, 96, 10565–10571. (30) Kwak, J.; Anson, F. C. Anal. Chem. 1992, 64, 250–256.

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How Do Structure and Charge Affect Metal ... - ACS Publications

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