In the Laboratory
Steady-State Fluorescence Anisotropy To Investigate Flavonoids Binding to Proteins
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Christine M. Ingersoll* and Christen M. Strollo Department of Chemistry, Muhlenberg College, Allentown, PA 18104; *
[email protected] Molecular fluorescence spectroscopy is an important topic covered in analytical or instrumental analysis courses and is used in experimental laboratories in other courses such as physical chemistry and biochemistry as well. Numerous fluorescence experiments for the undergraduate laboratory have been reported in this Journal,1 including laboratories based on specific applications of fluorescence such as fluorescence quenching (1–7) and time-resolved fluorescence techniques (1, 4, 8, 9). Of the fluorescence-based laboratory experiments published in this Journal, few (10–12) use fluorescence polarization or anisotropy to investigate the rotational mobility of the fluorophore. In fact, fluorescence polarization techniques, although useful for many applications, are not typically covered in the undergraduate chemistry curriculum. Here fluorescence anisotropy is used to investigate the binding of a model protein, human serum albumin, with the antioxidant quercetin, a flavonoid. The binding constant and Gibbs free energy change for the interaction are determined using both double reciprocal (linear) and nonlinear regression analyses. This laboratory experiment is wellsuited for students in third- and fourth-year chemistry courses, including instrumental analysis, physical chemistry, and biochemistry and can be completed within one threehour laboratory period.
time, fluorescence polarization measurements can be used to determine the rotational mobility of a fluorophore. Fluorescence anisotropy (r) is an experimental measure of fluorescence depolarization (the lower the anisotropy value, the faster the rotational diffusion; the higher the anisotropy, the slower the rotational diffusion). Anisotropy measurements are made by exciting the fluorophore with polarized light and measuring the fluorescence intensity both parallel and perpendicular to the excitation polarization (see schematic shown in Figure 2). Specifically, the sample is excited with vertically polarized light and the vertical and horizontal emission components (IVV and IVH) are measured. Anisotropy (r) is defined by the following equation (18) I − G I VH r = VV (1) I VV + 2G I VH where the G is the intensity ratio of the vertical (V) to horizontal (H) components of the emission when the sample is excited with horizontally polarized light (G = IHVIHH), which corrects for efficiency differences in the instrument optics (18). G is dependent on monochromator wavelength and slit widths; as long as the experimental conditions remain unchanged, G only needs to be measured once for the experiment.
Background Flavonoids, a large class of antioxidant polyphenolic compounds, have known health benefits with anti-inflammatory, anti-allergic, and even anti-tumor properties (13–16). Flavonoids are found naturally in plant material as secondary metabolites contributing to defense against herbivory (17) and in antioxidant-rich foods such as red wine, teas, berries, and onions (13, 14). Because flavonoids are known to bind to proteins in the body (16), it is important to understand the equilibrium constants and thermodynamics of such binding events to better understand their role in various organisms. Quercetin (3,3´,4´,5,7-pentahydroxyflavone; structure shown in Figure 1), like many other flavonoids, is inherently fluorescent, readily available, known to bind approximately 1:1 with serum albumins (14–16), and therefore appropriate as a model flavonoid. In this experiment, the binding of quercetin with human serum albumin (HSA) is investigated using steady-state fluorescence anisotropy. The binding constant (Kb) and the Gibbs free energy change (∆G ) of this interaction are determined from the anisotropy data.
Figure 1. Structure of quercetin.
Theory When a fluorescent molecule is excited with polarized light, the resulting fluorescence emission is also polarized. Since the main cause of fluorescence depolarization is rotational diffusion of the fluorophore during its excited-state lifewww.JCE.DivCHED.org
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Figure 2. Schematic of a fluorometer used to make polarization or anisotropy measurements: ex is excitation and em is emission.
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In the Laboratory
The measured anisotropy represents a weighted average of the anisotropies of the fluorophore if it is in multiple forms or environments. In this experiment, since two forms of quercetin are expected (free and protein-bound), the anisotropy is the weighted average of the anisotropies of free and bound quercetin (18) r = fF rF + fB rB (2) where fF and fB are the fractional fluorescence contributions of the free and bound forms of the fluorophore, respectively, and rF and rB are the corresponding anisotropy values. Because quercetin undergoes an increase in fluorescence intensity upon binding to HSA, a correction factor (R is the ratio of the intensities, IBIF, under the same monochromator settings as the anisotropy experiments) is incorporated. Rearranging eq 2 and including the intensity ratio, the fraction of bound quercetin is calculated as follows (18): r − rF fB = (3) R ( rB − r ) + r − rF Notice that if there is no change in intensity upon binding, R is equal to one and the equation reduces to a form of eq 2. Considering the equilibrium for the binding of quercetin to HSA,
Q + HSA
Q–HSA
the binding constant can be expressed as [ Q–HSA ] Kb = [Q ] [ HSA ] and the dissociation constant, Kd = 1Kb. The equilibrium expression is related to the fraction of quercetin bound by (18,19) [ HSA ] fB = (4) [HSA ] + K d or 1 1 1 = + 1 (5) K b [HSA ] fB This rearrangement (eq 5) allows for linear regression analysis via a double reciprocal plot and determination of the binding constant from the slope. In addition, a nonlinear regression analysis can be conducted fitting eq 4 directly to determine the binding constant. Experimental HEPES (N-2-hydroxyethylpiperazine-N´-2-ethanesulfonic acid), human serum albumin (essentially fatty acid free), and quercetin dihydrate were purchased from SigmaAldrich. A 50 mM, pH 7.0 HEPES buffer solution and two stock solutions of HSA (∼100 µM and ∼10 µM) in HEPES buffer are prepared in advance and stored in the refrigerator until the experiment. A 2 mM stock solution of quercetin in 95% ethanol is prepared fresh either prior to the lab or by the students at the start of the experiment. The quercetin solution is then diluted with HEPES buffer to 20 µM. A Varian Cary Eclipse fluorescence spectrophotometer with an automated polarizer accessory is used to perform the fluorescence anisotropy measurements, although inexpensive film polarizers can be used if the fluorometer is not already 1314
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Figure 3. As the concentration of HSA increases, the average fluorescence anisotropy, r, of quercetin increases initially and then levels off. Error bars represent ± one standard deviation of three measurements.
equipped with polarizers. The monochromator excitation and emission wavelengths are set to 370 nm and 530 nm, respectively, with corresponding 5 nm and 20 nm slit widths. All measurements are obtained with quartz cuvettes at a constant temperature of 25 ⬚C. Fluorescence anisotropy of free quercetin (12–15 µM, known accurately) is measured, followed by quercetin solutions with varying serum albumin concentrations up to 30 µM. The concentration of quercetin is kept constant by making up the volume with buffer in each cuvette. Fluorescence intensity measurements are performed on the free and bound quercetin solutions to obtain the intensity ratio of bound to free quercetin under the same wavelength and slit settings for data analysis. Hazards Quercetin dihydrate is toxic if swallowed; inhalation and contact with skin and eyes should be avoided. Human serum albumin is a potential biohazard and therefore, students should be warned of the risks associated with working with biohazards and should handle these solutions with extreme caution. It should be noted that the serum albumin protein used to make the solutions given to students in this experiment has tested negative for HIV and hepatitis B surface antigen. Although exposure to these and other infectious agents is highly unlikely, the solutions should be handled as if infectious agents are present. Minimally, appropriate protective eyewear, clothing, and heavy rubber gloves should be worn for this experiment. Results Typical student data for quercetin binding to HSA are shown in Figure 3.2 The quercetin anisotropy increases as protein is added and the fraction of bound quercetin (with a slower rotational diffusion) increases until it reaches a steady state value. The same data are then modeled two ways to determine the binding constant. First, the data are manipulated in the form of eq 5 to produce a linear relationship. This double reciprocal plot of 1fB as a function of 1[HSA], shown in Figure 4, is linear with a y intercept of 1 and a slope equal to the dissociation constant (Kd = 1Kb). The binding constant for these data is 1.91 × 105 L mol᎑1, and the Gibbs free energy change is calculated to be ∆G = ᎑30.1 kJ, consistent
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Figure 4. Double reciprocal plot, where the slope = Kd = 1/Kb.
Figure 5. Data and nonlinear least-squares fit to eq 4.
with a highly favorable protein–flavonoid interaction. Alternatively, the data are fit directly to eq 4 by constructing a plot of fraction bound as a function of HSA concentration, and the binding constant determined through nonlinear regression analysis.3 Figure 5 presents a binding isotherm showing the data and fit for the nonlinear regression analysis, resulting in a binding constant of 3.39 × 105 L mol᎑1 and ∆G of ᎑31.5 kJ. The binding constants determined with these data (and Kb values determined by other students in the lab) are in good agreement with those reported in the primary literature, ranging from 1.46 × 104 L mol᎑1 (20) to 5.30 × 105 L mol᎑1 (19), determined by various methods.
and other proteins (e.g., bovine serum albumin, BSA) have also been studied with the same method. 3. We use nonlinear regression software found online at http:/ /statpages.org/nonlin.html (accessed Apr 2007), although any nonlinear least-squares software can be used. The formula y = x(x + a) is analyzed where x = [HSA], y = fB, and a = Kd.
Conclusion This laboratory experience allows students to explore an aspect of fluorescence spectroscopy not typically covered in the undergraduate laboratory. Fluorescence polarization or anisotropy is used to determine the binding constant for a model flavonoid–protein interaction, an attractive system for students to work with owing to its relevance in the food and health industry and biochemistry. The experiment is interdisciplinary, exploring many aspects of chemistry such as equilibrium, thermodynamics, biochemical interactions, and data-analysis methods and can be completed in one threehour laboratory period. Acknowledgments The authors thank Thomas Betts of Kutztown University for his continuous input and the students in CHM 312 at Muhlenberg College. WSupplemental
Material
Instructions for students and notes for the instructor are available in this issue of JCE Online. Notes 1. In July 2006, a title search for “fluorescence” in the JCE Index online yielded 70 citations, and a keyword search for “FLUOR” in the Project Chemlab database resulted in over 100 laboratory experiments. 2. Data are shown here for the binding of quercetin to human serum albumin; however, other flavonoids (e.g., kaempferol) www.JCE.DivCHED.org
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