Sheila C. Wang and Deborah B. Zamble From the Department of Chemistry, University of Toronto, Ontario M5S 3H6, Canada

2006 by The International Union of Biochemistry and Molecular Biology BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION Printed in U.S.A. Vol. 34, No. 5, pp. 364 368, 2006 Laboratory Exercises Fluorescence Analysis of Sulfonamide Binding to Carbonic Anhydrase Received for publication, October 10, 2005, and in revised form, February 17, 2006 Sheila C. Wang and Deborah B. Zamble From the Department of Chemistry, University of Toronto, Ontario M5S 3H6, Canada A practical laboratory experiment is described that illustrates the application of fluorescence resonance energy transfer to the study of protein-ligand binding. The affinities of wild-type and mutant human carbonic anhydrase II for dansylamide were determined by monitoring the increase in ligand fluorescence that occurs due to energy transfer from tryptophan residues near the enzyme active site. In a subsequent experiment, the binding constant of azetazolamide, a weaker fluorophore but a stronger ligand, is measured by competition with dansylamide. This simple experiment introduces students to the widely used technique of fluorescence spectroscopy and to the determination of protein-ligand binding constants. Keywords: Fluorescence, fluorescence resonance energy transfer, ligand binding, carbonic anhydrase, sulfonamide. The interactions between proteins and ligands underlie many biological processes such as receptor signaling, antibody recognition, transcription, and enzymatic reactions. In these pathways, the ligands span a wide scope of biomolecules ranging from other proteins to small organic molecules or metal ions. One method that is often used to examine protein-ligand interactions is fluorescence, a sensitive and convenient technique for monitoring a variety of biomolecular transformations. Most proteins have an observable intrinsic fluorescence because of their aromatic residues, primarily tryptophan, and if the binding of a ligand alters the local environment of the fluorophores then a change in the intensity and/or maximum wavelength of fluorescence is observed. As an alternative, an external fluorescent probe or labeling reagent may be used to monitor ligand binding to proteins, provided that there is a detectable change in the surroundings of the fluorophore upon complex formation [1 5]. For background information on the principles of fluorescence and applications to biochemical systems, we refer readers to several resources [1, 2, 4], as well as a fluorescence tutorial web site (www.probes.invitrogen.com) hosted by Molecular Probes Inc. In this laboratory exercise, fluorescence is used to study ligand binding to human carbonic anhydrase II (hcaii). 1 Carbonic anhydrase (CA) catalyzes the reversible hydration of carbon dioxide to produce a bicarbonate anion and a proton (Reaction 1). To whom correspondence should be addressed: Dept. of Chemistry, University of Toronto, 80 St. George Street, Toronto, Ontario M5S 3H6, Canada. E-mail: dzamble@chem.utoronto.ca. 1 The abbreviations used are: CA, carbonic anhydrase; hcaii, human CA II; AZ, acetazolamide; DNSA, dansylamide; FRET, fluorescence resonance energy transfer. 364 CO 2 H 2 O 7 HCO 3 H REACTION 1 By virtue of this catalytic activity, carbonic anhydrase plays an important role in multiple physiological processes including CO 2 homeostasis and gas transport between tissues and lungs [6, 7]. Multiple structurally homologous cytosolic isozymes of carbonic anhydrase are known to occur in higher vertebrates [8, 9], and of these enzymes, hcaii is probably the most extensively characterized by both mechanistic and high resolution structural studies [7, 9, 10]. Carbonic anhydrase enzymes are inhibited by sulfonamides [11], and for hcaii micromolar to nanomolar dissociation constants of several of these inhibitors have been reported [12]. The small dissociation constants observed for the aromatic sulfonamide inhibitors may be due to their ability to bind to hcaii through two groups: a hydrophobic interaction between the aromatic structures and a hydrophobic patch in the protein as well as a bond between the sulfonamide moiety and the zinc ion at the catalytic active site [12 15]. Several sulfonamides are used in the clinic [7]; thus, the study of sulfonamide binding to hcaii is of both scientific and medical interest. Furthermore, this experiment illustrates clearly that fluorescence, and specifically fluorescence resonance energy transfer (FRET), is a very sensitive method for obtaining the dissociation constants of strong interactions and permits titrations of solutions containing low concentrations (10 7-10 6 M) of protein. In this laboratory exercise, hcaii and sulfonamide inhibitors are used to illustrate the application of fluorescence, and in particular FRET, to monitor protein-ligand binding and to measure the affinity constants. We propose two experiments to the students. The first is a fluorescence This paper is available on line at http://www.bambed.org

365 assay to measure the affinity of hcaii for the fluorophore dansylamide (DNSA). When DNSA binds, there is an increase in the fluorescence emission intensity [13] due to FRET from the tryptophan residues near the active site, as well as a blue shift in the wavelength of maximum emission. It is therefore possible to directly measure the submicromolar DNSA dissociation constant by monitoring the enhanced fluorescence that occurs as DNSA is titrated into a protein solution. In the second part of the experiment, students measure the dissociation constant of acetazolamide (AZ), an inhibitor of hcaii that binds at the same site as DNSA. AZ is not a bright fluorophore, and it binds very tightly to hcaii with a dissociation constant of 9.6 nm for wild-type enzyme [12]. Thus, the affinity of AZ would be difficult to measure directly. Instead, the binding of AZ is assayed in a competition with DNSA. By using the DNSA dissociation constant that was measured in the previous experiment, the students can then calculate the AZ dissociation constant. EXPERIMENTAL PROCEDURES Time and Teams These experiments were performed in a 4-h block by students working in teams of two. The students worked in pairs because of a limiting number of fluorescence spectrophotometers. The success of this experiment depends on accurate pipetting and careful mixing, so it is beneficial if the students have prior experience with using micropipettes. Materials Wild-type and mutant hcaii were expressed and purified by the students in a previous laboratory exercise. The expression vector was generously supplied by D. N. Silverman [16], and the protein was purified by using a single affinity chromatography step as described [17]. Dansylamide, acetazolamide, and Me 2 SO were purchased from Aldrich. Solutions The solutions used were 20 mm Tris buffer, ph 8.0, 10 mm dansylamide stock in Me 2 SO, 10 mm acetazolamide stock solution in Me 2 SO, and 0.25 mm hcaii in 20 mm Tris, ph 8.0. The 10 mm DNSA and AZ stocks were each diluted to 1 and 0.2 mm in Me 2 SO, respectively. By making several additional dilutions into buffer, the amount of Me 2 SO added to the protein is minimized. Spectroscopic Measurements Fluorescence measurements were performed at 25 C by using a PerkinElmer Life Sciences LS-50B fluorometer. Each fluorescence spectrophotometer should be set up according to the appropriate operating instructions for that instrument. The excitation wavelength was set to 280 nm, and the scan speed was set to 100 nm/min. Students first scanned the emission wavelength of the buffer from 400 to 520 nm to confirm that there were no fluorescent contaminants. Students measured the fluorescence intensity at 470 nm of a sample containing 0.25 M hcaii and 20 M DNSA, a saturating concentration, and set the fluorescence intensity of this sample to 50 80% of the instrument range by appropriate adjustment of the slit widths. These parameters were used for the entire experiment. If time permits, the students should also scan the protein alone and monitor 200 600 nm so that they can observe the tryptophan fluorescence at 336 nm and how weak it is in intensity. Furthermore, both excitation and emission scans of DNSA would reveal that the excitation spectrum overlaps with the emission spectrum of the tryptophans. Samples DNSA binding was assayed by titrating small increments of DNSA (1 5 l) into 1-ml samples containing 0.25 M hcaii in 20 mm Tris, ph 8.0. AZ binding was assayed in competition with bound DNSA. Small increments of AZ (1 5 l) were titrated into 1-ml samples containing 0.25 M hcaii, 20 mm Tris, ph 8.0, and 20 M DNSA. After each titration, the solution was mixed gently by pipetting up and down. The samples were allowed to equilibrate at room temperature for 1 min before scanning. DATA ANALYSIS Adjust Fluorescence for Dilution The observed fluorescence is adjusted for dilution by using Equation 1. Fl adj Fl obs Vol fin /Vol ini (Eq. 1) where Fl adj is the adjusted fluorescence, Fl obs is the observed fluorescence, Vol fin is the final volume, and Vol ini is the initial volume. This adjustment normalizes the fluorescence to constant volume and protein concentration. Calculate the DNSA Dissociation Constant The dissociation constant, K D, is defined by Equation 2, K D P DNSA (Eq. 2) P-DNSA where [P] is the concentration of free protein, [DNSA] is the concentration of free ligand, and [P-DNSA] is the concentration of the protein-ligand complex. In many cases, these types of experiments are performed with a protein concentration far below the K D,so we may assume that [L] [L] T, where [L] T is the total ligand concentration. However, in this case, we are working at a [P] close to the K D, so we can not make that assumption. Instead, the total ligand concentration is described by Equation 3. DNSA T DNSA P-DNSA (Eq. 3) The concentration of the complex, [P-DNSA], is calculated at each data point from Equation 4. r P-DNSA F adj F min (Eq. 4) P T F max F min where r is the fraction saturation of hcaii with DNSA, F min is the fluorescent signal with no ligand added, F max is the fluorescent signal at saturation, and F adj is the adjusted fluorescent signal observed at each concentration of ligand. Calculate the fraction saturation, r, for each data point and then [P-DNSA] and [DNSA]. Plot the data as [DNSA] versus r. Fit this curve to the Langmuir isotherm (Equation 5) and determine the dissociation constant, K DNSA, for DNSA-binding to hcaii. DNSA r (Eq. 5) K DNSA DNSA Calculate AZ Dissociation Constant Again, the fluorescence needs to be adjusted for dilution first by using Equation 1. In this experiment, the concentration of DNSA is much higher than the concentration of hcaii, so we can assume that [DNSA] T [DNSA]. Furthermore, because AZ binds tightly to hcaii, we can assume that any protein that does not have DNSA bound has AZ bound or, in other words, there is no free protein. Again, r is fractional saturation of hcaii with DNSA, and it is calculated with Equation 4. Therefore and P-AZ P T 1 r (Eq. 6) AZ AZ T P-AZ (Eq. 7)

366 BAMBED, Vol. 34, No. 5, pp. 364 368, 2006 FIG. 1.Fluorescence titration of hcaii with DNSA. Emission spectra ( exc 280 nm) of wild-type hcaii (0.25 M) titrated with increasing concentrations of DNSA (0, 0.1, 0.3, 1.4, 4, 10, and 17 M) are shown. At higher concentrations of DNSA, the intensity at 470 nm remained constant but increased at longer wavelengths, probably due to the Me 2 SO. Plot r versus [AZ] and fit the data to the following equation (Equation 8) to calculate the affinity of HCAII for AZ. 1 r (Eq. 8) 1 K DNSA / DNSA 1 AZ /K AZ RESULTS Binding of DNSA by Human Carbonic Anhydrase II The binding of DNSA by hcaii is accompanied by an enhancement of the ligand fluorescence emission at 470 nm (Fig. 1). It can be observed, therefore, that DNSA is a good fluorescent probe of the sulfonamide-binding site of hcaii. A general prerequisite for energy transfer to occur is that the emission band of the donor molecule overlaps the absorption band of the acceptor molecule [18]. This prerequisite is met in the hcaii-dnsa system because the protein fluorescence band ( max 336 nm) strongly overlaps the DNSA absorption band [13]. The probability of energy transfer between two molecules is dependent on the inverse of the sixth power of the intermolecular distance. In practice, the effective intermolecular separation range is 10 100 Å, comparable with the dimensions of biological macromolecules. The strong fluorescence enhancement suggests that the sulfonamide must be a close distance to at least one of the seven tryptophans in hcaii. DNSA Binding Affinity The dissociation constants for DNSA binding to hcaii variants were measured by monitoring the fluorescence increase at 470 nm with increasing concentrations of DNSA until saturation was observed (Fig. 2). The data fit well to the Langmuir isotherm and may therefore be described by a single observable dissociation constant. The K DNSA for wild-type hcaii was determined to be 0.2 0.1 10 6 M (Table I, the average of data from five student teams). This value is in reasonable agreement with the K DNSA of 0.93 10 6 M reported previously by Nair et al. [12]. AZ Binding Affinity The dissociation constants for AZ were measured in competition with DNSA by monitoring the decrease in fluorescence due to the disappearance of the hcaii DNSA complex and the concomitant formation of a hcaii AZ complex (Fig. 3). The K AZ for wild-type hcaii was determined to be 6 3 10 9 M (Table I, the average of data from five student teams). Again, this value is in FIG. 2.Determination of DNSA dissociation constants. Small aliquots (1 5 l) of DNSA were added to 1 ml of 0.25 M HCAII, and the fluorescence emission was monitored at 470 nm with ex 280 nm. Data for both wild-type hcaii (filled diamonds, solid line) and the E106A mutant (open diamonds, dashed line) are shown. The lines represent the fit of the data to Equation 5 by using Excel Solver. TABLE I Dissociation constants for DNSA and AZ binding to hcaii Protein K DNSA a M K AZ a Wild type 0.2 0.1 (5) 6 3 (5) E106A 0.8 0.2 (2) 9 1 (2) E106D 0.2 (1) 4 (1) A65F 0.002 (1) 3 (1) T199A 0.04 (1) 0.002 (1) a K DNSA and K AZ values were calculated by fitting the data to Equations 5 and 8, respectively, using Excel Solver. Errors are listed as standard deviations, and the number of independent experiments performed by student teams is indicated in parentheses. FIG. 3. Determination of AZ dissociation constants. Small aliquots (1 5 l) of AZ were added to 1 ml of 0.25 M hcaii and 20 M DNSA and the fluorescence emission was monitored at 470 nm with ex 280 nm. Data for both wild-type hcaii (filled diamonds, solid line) and the E106A mutant (open diamonds, dashed line) are shown. The lines represent the fit of the data to Equation 8 by using Excel Solver. good agreement with the K AZ of 9.6 10 9 M, reported previously [12]. Effect of Mutations on Sulfonamide Binding Amino acid substitution at Glu-106, Ala-65, and Thr-199 only mildly affect the binding of the two inhibitors, DNSA and AZ (Table I). For example, mutation of the residue Glu-106, which is involved in the active site [10], to an alanine decreases the binding affinities for DNSA and AZ by only 4- and 1.5-fold, respectively (Figs. 2 and 3 and Table I). Overall, the mutants studied in the class bind DNSA and AZ with affinities comparable with wild type. DISCUSSION OF THE DATA The data from wild-type hcaii and all of the mutants studied were compiled by several teams and presented to nm

367 FIG. 4. Image of AZ bound the active site of hcaii. This student-generated image was used in a presentation to illustrate the ligand bound in the catalytic active site of the protein. The nearby tryptophans are not shown. This figure was prepared by using Deep View molecular viewing software. the entire class in a seminar format. The students discussed how the experiments were performed and analyzed and compared the class data with the values that have been reported in the literature. Furthermore, the comprehensive literature on hcaii allowed for a more in-depth analysis. For example, students also prepared pictures from the available crystal structures of AZ bound to the active site of hcaii (Fig. 4), and they discussed how this structure supports a stoichiometric binding model. The tryptophans in the secondary coordination sphere around the zinc were highlighted by the students and identified as the source of the FRET. The structures are also useful for the discussion about the mutants, and the students hypothesized why or why not the mutations affected sulfonamide binding. CONCLUDING REMARKS This laboratory exercise is designed to teach students about protein-ligand binding and to introduce them to fluorescence as a powerful tool for measuring biomolecular interactions. The lectures that accompany the laboratories include discussions of the theory behind fluorescence and FRET, the importance of ligand binding in nature, the practical requirements for measuring dissociation constants, and the analysis of more complicated systems. This experiment was used in the context of a third year biological chemistry course that examines the structure-function relationship of hcaii through a series of experiments with wild-type and mutant enzyme. The affinities of these sulfonamide inhibitors have not been reported for all hcaii mutants, so this laboratory exercise has the added advantage of allowing students to contribute new information to an active field of research. In addition, AZ was also examined as an inhibitor in steady-state kinetics experiments, so the students could compare the K D they measured by fluorescence with the K I measured in the kinetics experiment. However, this experiment could be used in a laboratory course with less integration of the experiments, and performed with bovine carbonic anhydrase [13], which is FIG. 5.DNSA and AZ dissociation constants. DNSA and AZ were titrated into hcaii, and the data were fit as described in the legends for Figs. 2 and 3 to generate a K DNSA of 0.2 M and K AZ of 4 nm. These data were generated by the graduate student teaching assistant (S. C. W.). commercially available or easily purified from bovine blood [17]. In such a course, some additional information could be presented by the instructor, such as pictures of AZ bound to the active site and a description of AZ as an inhibitor. Acknowledgments We thank Prof. Carol Fierke for permission to design this experiment based on Dr. Fierke s experimental approach and for giving a talk about research on carbonic anhydrase at the University of Toronto, which put this laboratory exercise into a greater context for the students that year. We thank Tyler Lougheed, the joint laboratory demonstrator, for assisting the students and for helpful discussions. We thank the CHM379 students for their enthusiastic participation and positive feedback, especially Carol Wang for giving us a figure of AZ bound to hcaii. REFERENCES [1] D. Freifelder (1982) in Physical Biochemistry, pp. 537 572 (Wilson, J., ed.) W. H. Freeman and Company, New York. [2] J. R. Lakowicz (1983) Principles of Fluorescence Spectroscopy, pp. 341 379, Plenum Publishing Corp., New York. [3] A. Marty, M. Boiret, M. Deumie (1986) How to illustrate ligand-protein binding in a class experiment, J. Chem. Ed. 63, 365 366. [4] J. C. Croney, D. M. Jameson, R. P. Learmonth (2001) Fluorescence spectroscopy in biochemistry: Teaching basic principle with visual demonstrations, Biochem. Mol. Biol. Ed. 29, 60 65. [5] M. Möller, A. Denicola (2002) Study of protein-ligand binding by fluorescence, Biochem. Mol. Biol. Ed. 30, 309 312. [6] R. P. Henry (1996) Multiple roles of carbonic anhydrase in cellular transport and metabolism, Annu. Rev. Physiol. 58, 523 38. [7] S. Lindskog (1997) Structure and mechanism of carbonic anhydrase, Pharmacol. Ther. 74, 1 20. [8] D. N. Silverman, S. Lindskog (1988) The catalytic mechanism of

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