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

F 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 experi-
ments 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 instrumen-
tation. 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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Journal of Chemical Education
Figure 2. DNA double helix intercalated by two proflavine molecules
(in spheres).
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 neon-
green 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.
■ 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 3. SpectroVis Plus device connected to a handheld Vernier
LabQuest computer (used with permission from Vernier).
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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.
■ 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.
■ 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.
Figure 4. Normalized absorbance and fluorescence spectra of
proflavine.
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.
Journal of Chemical Education
Figure 5. Proflavine fluorescence as a function of DNA concentration
in the absence (open circles) and presence (filled triangles) of 100
mM NaCl.
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.
■ 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 (fluo-
rescence) and important molecular phenomenon (drug DNA
binding) has been made accessible and exciting to first-year
chemistry students.
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■
Laboratory Experiment
ASSOCIATED CONTENT
*
S Supporting Information
Detailed student handout and instructor notes. This material is
available via the Internet at http://pubs.acs.org.
■ AUTHOR INFORMATION
Corresponding Author
*E-mail: sjain@bard.edu.
Notes
The authors declare no competing financial interest.
■ 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.
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