Laboratory Experiment pubs.acs.org/jchemeduc
Proflavine−DNA Binding Using a Handheld Fluorescence Spectrometer: A Laboratory for Introductory Chemistry Swapan S. Jain,* Christopher N. LaFratta, Andres Medina, and Ian Pelse Department of Chemistry, Bard College, Annandale-on-Hudson, New York 12504, United States S Supporting Information *
ABSTRACT: Fluorescence spectrometers are expensive instruments and fluorimetry is rarely used in undergraduate teaching curricula, especially in the first year. The experiment described utilizes handheld spectrometers capable of measuring absorbance and fluorescence to introduce students to drug binding to DNA. Students monitor the changes that occur in fluorescence intensity when proflavine binds to DNA in the presence and absence of sodium ions. The goals of this experiment are to illustrate principles of electronic structure, noncovalent interactions, and spectroscopic techniques. The handheld device utilized in this experiment is inexpensive and the ease of operation makes fluorescence spectroscopy accessible and exciting to first-year undergraduates.
KEYWORDS: First-Year Undergraduate/General, Analytical Chemistry, Biochemistry, Laboratory Instruction, Hands-On Learning/Manipulatives, Noncovalent Interactions, Nucleic Acids/DNA/RNA, Fluorescence Spectroscopy, Drugs/Pharmaceuticals
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luorescence spectroscopy has been widely used in research and practical applications as an analytical tool.1 Its use is on the rise especially because researchers are moving away from deploying hazardous chemicals and radioisotopes for analysis. Fluorescence is a form of spectroscopy whereby emission of light from an electronically excited state occurs after absorption of higher frequency light.2 Fluorescence spectroscopy has generally been introduced to advanced biochemistry students in the undergraduate curriculum.3 Many of the previous experiments using fluorimetry use carcinogens, such as ethidium bromide.4,5 Furthermore, these experiments use bulky and expensive instruments that are not viable for large classes typical in the introductory chemistry setting.6 In this laboratory experiment, the binding of proflavine molecule to DNA is detected using handheld fluorimeters. The total cost of the instrumentation is less than $800. The pedagogical goals of this experiment were to illustrate concepts such as electronic structure, intermolecular forces, and pi bonding, all of which are taught during the first semester of an introductory chemistry course. In this experiment, students take advantage of commercially available, low-cost instrumentation to explore the molecular structure of proflavine and its binding to DNA using spectroscopy. Proflavine (Figure 1) is in the acridine family of fluorescent dyes.7 It has an excitation wavelength of 444 nm and an emission wavelength of 520 nm. It is a planar, heterocyclic molecule that interacts with DNA by intercalation (Figure 2). Intercalation is a binding mode whereby small, conjugated molecules insert themselves between the base pairs of double helical DNA.8 This type of a binding mode is favorable due to © XXXX American Chemical Society and Division of Chemical Education, Inc.
Figure 1. Chemical structure of proflavine at pH 7.
the pi−pi stacking between proflavine and the adjacent base pairs. Intercalation alters DNA structure by unwinding and lengthening the double helical DNA. Many pharmaceutical compounds (e.g. doxorubicin, daunomycin, actinomycin) available on the market today interact with DNA by an intercalative binding mode.9 Molecules, such as proflavine, follow the nearest neighbor exclusion principle when binding to double stranded DNA. This means that intercalators can bind to a maximum occupancy of one molecule every two base pairs of DNA.10 At pH 7, proflavine, which has a pKa of 8.2, is predominately in the cation form. Previous work has shown that positively charged proflavine molecules can stack alongside the negatively charged phosphate backbone, but the preferred mode of proflavine binding to DNA at pH 7 is intercalation.11 A fluorescence experiment is described where first-year students are introduced to DNA−drug binding in a straightforward manner using relatively inexpensive instrumentation. The excited state of proflavine is quenched upon binding to DNA, which means that its fluorescence intensity decreases. Thus, the change in the fluorescence of proflavine offers a window through which its binding with DNA can be monitored. Students also investigate the effect of cations,
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dx.doi.org/10.1021/ed300481u | J. Chem. Educ. XXXX, XXX, XXX−XXX
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Laboratory Experiment
titrated with a 60 μM per base pair DNA stock solution. The DNA solution was prepared in 1 μM proflavine, which keeps the proflavine concentration unchanged over the course of the titration. In a separate titration, 1 μM proflavine solution containing 100 mM NaCl was titrated with a 60 μM per base pair DNA solution prepared in 1 μM proflavine and 100 mM NaCl. Students work in pairs and collect 12 data points for each titration. On average, students take ∼1 h to finish collecting data during this experiment. Additional details regarding preparation of samples and the procedure are provided in the Supporting Information.
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HAZARDS Exposure to proflavine may cause respiratory inflammation, irritation of skin, and irritation of eyes. It should be stored as a harmful chemical waste and disposed according to standard chemical safety and hygiene protocols.
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Figure 2. DNA double helix intercalated by two proflavine molecules (in spheres).
RESULTS AND DISCUSSION Students measured the absorption of a proflavine solution (∼25 μM) and accurately determined its concentration using the Beer−Lambert law using the extinction coefficient of proflavine (see Experimental Details). Proflavine at 1 μM concentration was excited at 405 nm and the resulting emission spectrum was recorded as a function of wavelength with peak emission occurring at a wavelength of ∼520 nm. Representative absorption and fluorescence emission spectra of proflavine (student data) are shown in Figure 4.
such as sodium, on the binding of proflavine to DNA. This experiment can be performed in a 2-h lab period. More than 180 first-semester general chemistry students during three semesters (fall 2010−2012) have carried out minor variations of this experiment. Students were captivated with the neongreen fluorescence of proflavine and its subsequent quenching following the addition of DNA. Furthermore, the excitement and ease of operation afforded by handheld spectrometers was clearly evident in the lab classes.
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EXPERIMENTAL DETAILS Proflavine and DNA (from salmon testes) solutions were prepared in 10 mM sodium phosphate buffer (pH 7.1). Concentrations were checked using UV−vis spectroscopy. Proflavine has a peak absorbance at 444 nm where ε444 = 38,900/(M cm).12 DNA stock solution was quantified where OD260 of 1.0 for double-stranded DNA = 50 μg/mL solution.13 Laboratory personnel prepare and test the concentrations of solutions in advance. Stock solutions of proflavine were titrated with DNA at room temperature and the accompanying changes in fluorescence were measured using a handheld spectrometer (a Vernier instrument equipped with SpectroVis Plus) (Figure 3). In a typical titration, 1 μM proflavine stock solution was
Figure 4. Normalized absorbance and fluorescence spectra of proflavine.
Figure 3. SpectroVis Plus device connected to a handheld Vernier LabQuest computer (used with permission from Vernier).
During fluorescence titration of proflavine with DNA, small increments of 60 μM per base pair double stranded DNA were titrated into a cuvette containing 1 μM proflavine. The stock DNA solution also contained 1 μM proflavine so that the proflavine is not diluted during the titration. A typical student plot of fluorescence intensity as a function of DNA concentration is shown in Figure 5. An exponential decline in fluorescence intensity of proflavine is observed upon addition of DNA (open circles in Figure 5). At high concentrations of DNA (far right on the x-axis), the bulk of proflavine molecules present in the solution are intercalated between the base pairs of DNA. Proflavine concentration is constant at 1 μM during the course of the titration. The final concentration of DNA is ∼18 μM which is in excess of what is required for proflavine to fully intercalate DNA according to the nearest neighbor principle.10 The absence of free proflavine in solution at highest DNA concentration leads to a near complete absence of fluorescence intensity. B
dx.doi.org/10.1021/ed300481u | J. Chem. Educ. XXXX, XXX, XXX−XXX
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ASSOCIATED CONTENT
S Supporting Information *
Detailed student handout and instructor notes. This material is available via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS Bard Summer Research Institute provided generous funding for this work. We are also grateful to the students who conducted this experiment in our general chemistry labs.
Figure 5. Proflavine fluorescence as a function of DNA concentration in the absence (open circles) and presence (filled triangles) of 100 mM NaCl.
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Binding of cationic intercalators such as proflavine to DNA occurs not only due to the stacking interactions between proflavine rings and base rings of DNA but also due to the electrostatic interactions between positively charged proflavine and the negatively charged phosphate backbone of DNA.14 Previous work has shown that the presence of cations, such as sodium and magnesium, lowers the binding of cationic intercalators to DNA.14 Electrostatic interactions between cations and negatively charged phosphate groups in DNA decreases the binding of cationic proflavine to DNA. Students investigated this phenomena using a sample containing 1 μM proflavine and 100 mM NaCl. DNA was, again, titrated incrementally into the cuvette. The titrated DNA solution also contained proflavine and NaCl in order to keep proflavine and sodium ion concentration constant during the titration. Similar to the previous titration, a smooth and exponential decline in fluorescence intensity was observed as a function of increasing DNA concentration (filled triangles in Figure 5). This observation indicated that binding of proflavine to DNA was diminished in the presence of sodium ions. The data obtained by students could be used to determine equilibrium binding constants using nonlinear least-squares fitting methods as described previously.15,16 However, the model fitting exercise is more suited for upper-level chemistry students with an understanding of thermodynamics, equilibrium association constants, and statistical analysis.
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CONCLUSIONS During the last three years, undergraduate students have investigated proflavine binding to DNA using fluorescence spectroscopy. Students have consistently obtained data that shows a significant decline in fluorescence intensity when proflavine binds to DNA and a lesser decline in fluorescence intensity when the titration was conducted in the presence of sodium ions. This experiment has allowed students to gain practical exposure in absorption and fluorescence spectroscopy, understanding of molecular structure, importance of pi stacking, charge−charge interactions, and DNA binding to drugs. Students write a detailed lab report and are also tested on topics related to the experiment on the subsequent lecture exam. With the use of inexpensive and easy to use instrumentation, an advanced experimental technique (fluorescence) and important molecular phenomenon (drug DNA binding) has been made accessible and exciting to first-year chemistry students. C
dx.doi.org/10.1021/ed300481u | J. Chem. Educ. XXXX, XXX, XXX−XXX
