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Pro Avine DNA Binding Using A Handheld Fluorescence Spectrometer: A Laboratory For Introductory Chemistry
Pro Avine DNA Binding Using A Handheld Fluorescence Spectrometer: A Laboratory For Introductory Chemistry
pubs.acs.org/jchemeduc
■ 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.
Figure 2. DNA double helix intercalated by two proflavine molecules
(in spheres). ■ RESULTS AND DISCUSSION
Students measured the absorption of a proflavine solution (∼25
such as sodium, on the binding of proflavine to DNA. This μM) and accurately determined its concentration using the
experiment can be performed in a 2-h lab period. More than Beer−Lambert law using the extinction coefficient of proflavine
180 first-semester general chemistry students during three (see Experimental Details). Proflavine at 1 μM concentration
semesters (fall 2010−2012) have carried out minor variations was excited at 405 nm and the resulting emission spectrum was
of this experiment. Students were captivated with the neon- recorded as a function of wavelength with peak emission
green fluorescence of proflavine and its subsequent quenching occurring at a wavelength of ∼520 nm. Representative
following the addition of DNA. Furthermore, the excitement absorption and fluorescence emission spectra of proflavine
and ease of operation afforded by handheld spectrometers was (student data) are shown in Figure 4.
clearly evident in the lab classes.
■ 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 Figure 4. Normalized absorbance and fluorescence spectra of
3). In a typical titration, 1 μM proflavine stock solution was proflavine.
■
Laboratory Experiment
ASSOCIATED CONTENT
*
S Supporting Information
■ AUTHOR INFORMATION
Corresponding Author
*E-mail: sjain@bard.edu.
Notes
The authors declare no competing financial interest.
■ CONCLUSIONS
During the last three years, undergraduate students have
with Nucleic Acids. Biopolymers 1968, 6, 1225−1253.
(12) Jain, S. S.; Stahle, C.; Anet, F.; Hud, N. V. Enzymatic Behavior
by Intercalating Molecules in a Template-Directed Ligation Reaction.
Angew. Chem., Int. Ed. 2004, 43, 2004−2008.
investigated proflavine binding to DNA using fluorescence (13) Promega Corporation Online Calculator. http://www.promega.
spectroscopy. Students have consistently obtained data that com/techserv/tools/biomath/calc07.htm. (accessed Jun 2013).
shows a significant decline in fluorescence intensity when (14) Drummond, D. S.; Gildemeister, V. F. W.; Peacocke, A. R.
proflavine binds to DNA and a lesser decline in fluorescence Interaction of Aminoacridines with Deoxyribonucleic Acids: Effects of
intensity when the titration was conducted in the presence of Ionic Strength, Denaturation, and Structure. Biopolymers 1965, 3,
sodium ions. This experiment has allowed students to gain 135−153.
practical exposure in absorption and fluorescence spectroscopy, (15) Qu, X.; Chaires, J. B. Analysis of Drug-DNA Binding Data.
understanding of molecular structure, importance of pi Methods Enzymol. 2000, 321, 353−369.
stacking, charge−charge interactions, and DNA binding to (16) Horowitz, E.; Hud, N. V. Ethidium and Proflavine Binding to a
2′,5′-Linked RNA Duplex. J. Am. Chem. Soc. 2006, 128, 15380−15381.
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 (fluo-
rescence) 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