Complementary Spectroscopic Assays for Investigating Protein

In the Laboratory edited by

Joseph J. BelBruno Dartmouth College Hanover NH 03755

Complementary Spectroscopic Assays for Investigating Protein-Ligand Binding Activity: A Project for the Advanced Chemistry Laboratory David P. Mascotti and Mark J. Waner* Department of Chemistry, John Carroll University, University Heights, Ohio 44118 *[email protected]

Protein-ligand interactions are important to many biological processes and experiments examining these interactions have received some attention in this Journal (1-3). One of the more traditional protein-ligand binding assays done in undergraduate biochemistry laboratories involves the examination of the streptavidin or avidin interaction with biotin (4). Traditionally, this has been done by the displacement of 2-(4-hydroxyphenylazo)benzoic acid (HABA), which is a weakly binding biotin analog (5, 6). Though still widely used, this assay requires relatively high quantities of protein (7). Because of the importance of the streptavidin-biotin system to biotechnology applications, a wide variety of other assays with varying sensitivities have also been developed (8-17). These assays utilize a variety of techniques, often using functionalized protein or biotin. Recently, Kada et al. synthesized biotin-4-fluorescein (B4F) as a highly sensitive fluorescent ligand for quantitating binding to avidin and streptavidin (SA) (11, 18). When bound to streptavidin, the fluorescence signal of the fluorescein is highly quenched (g80%). In a recently published article, we have shown that a significant portion of this fluorescence quench is due to a decrease in the extinction coefficient of the fluorescein moiety (19). This phenomenon serves as the basis of a new spectrophotometric assay that complements the fluorescence assay of Kada et al. This system of SA and B4F serves as the basis for a guidedinquiry laboratory project carried out during two, 4-h laboratory periods with an additional 1-h discussion. This project involves student refinement of general procedures and assessment of different techniques and assays, in addition to the traditional measurement of protein-ligand binding. While carrying out the experiment, students must demonstrate proper pipetting, estimation, titration, and experimental-design skills. This project fits into our biochemistry laboratory course, which has an analytical laboratory as a prerequisite and physical chemistry as a pre- or co-requisite. This project could also fit well in a biophysical or advanced laboratory course. Experimental Procedure Reagents and Equipment The solvent for all solutions was 10 mM triethanolamine (TEA) at pH = 7.3 prepared using Type I, 18 MΩ cm water. Streptavidin stock solutions were prepared from lyophilized protein (Prozyme, San Leandro, CA) with a nominal concentration of 140 μM (∼7 mg/mL). The actual SA concentration was determined using spectrophotometric analysis at A280 with ε2801mg/mL = 3.2 cm-1 (20). The nominal stock D-biotin

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(Fluka, Buchs, Switzerland and Sigma-Aldrich, St. Louis, MO) concentration was 1.8 mM, and the nominal stock biotin-4-fluorescein (Invitrogen, Carlsbad, CA) was 33 μM. The biotin and B4F solution concentrations are standardized via independent fluorescence titrations. Spectrophotometry was performed using a Varian Cary50 UV-vis and ThermoSpectronic Genesys 20 visible spectrophotometers, using disposable semimicro cuvettes. Fluorescence spectroscopy was carried out using a Photon Technology International (Lawrenceville, NJ) QuantaMaster dual-emission spectrofluorimeter with a single, stirred sample cell. Excitation illumination at 493 or 280 nm was provided by a 75 W xenon arc lamp. Bandpasses were 2 and 8 nm for excitation and emission, respectively. Data were collected in time-based emission mode averaging over 4 s (1 s per point). Solutions were equilibrated in the instrument with magnetic stirring for a minimum of 2 min after each addition of titrant. When not collecting data, the excitation shutter was closed to avoid photobleaching. When measuring B4F fluorescence, the photomultiplier tube (PMT) voltages were 810 V with a gain of 10-3, with 493 nm excitation and 520 nm emission. When measuring SA fluorescence, the PMT voltages were 930-950 V with a gain of 1, with 280 nm excitation and 350 nm emission. Assay Protocols During the first week, a “crude” titration of SA into 1 mL of 0.6-0.8 μM B4F was performed by all students using benchtop visible spectrophotometers. Small aliquots of a diluted SA solution were titrated into the B4F to determine the breakpoint in the absorbance signal at 493 nm. Students were asked to estimate the SA concentration and aliquots necessary to do this, given the four binding sites of the SA tetramer. They were told the goal is to observe saturation within about 6-8 aliquots. This typically took students approximately 2-3 h. During the second week, students worked in pairs and were asked to refine this experiment, optimizing to get 10-12 points with multiple points before and after the observed breakpoint. Each group of students then obtained data using either fluorescence or UV-vis spectrophotometers to examine forward (B4F into SA) or reverse titrations (SA into B4F). Additionally, one group performed a titration of biotin into SA utilizing intrinsic SA tryptophan fluorescence. The second week took from 2 to 4 h, limited by the availability of a single fluorimeter and Cary 50 UV-vis spectrophotometer. Additional information for each assay protocol is provided in the supporting information.

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r 2010 American Chemical Society and Division of Chemical Education, Inc. pubs.acs.org/jchemeduc Vol. 87 No. 7 July 2010 10.1021/ed100199j Published on Web 05/04/2010

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Journal of Chemical Education

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In the Laboratory

Figure 1. Spectrophotometric binding assay data: (A) titration of B4F into SA (forward titration; the light gray line is the fit to all data points) and (B) titration of SA into B4F (reverse titration).

Figure 2. Fluorescence binding assay data (λex = 493 nm, λem = 520 nm): (A) titration of B4F into SA (forward titration) and (B) titration of SA into B4F (reverse titration).

Hazards Biotin-4-fluorescein, biotin, and streptavidin may be harmful if inhaled, absorbed through skin, or swallowed and may cause irritation to skin, respiratory tract, and eyes. Triethanolamine has delayed target organ (liver, kidney) effects, may be harmful if inhaled, absorbed through skin, or swallowed and causes irritation to skin, respiratory tract, and eyes. Gloves should be worn when working with solids or solutions of these chemicals. Results Typical results obtained using the Cary 50 UV-vis spectrophotometer to examine forward and reverse titrations are shown in Figure 1. Note that in all cases the independent variable was the titrant-to-analyte concentration ratio. A clear breakpoint at stoichiometric binding was observed, whether using the Cary 50 or the Genesys 20 spectrophotometer. In each case, students were asked to perform linear regression on the two data regions, which allowed the students to determine the uncertainty in the observed equivalence 736


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point. So as not to bias data near the breakpoint, students were instructed to omit the two points closest to the apparent breakpoint. The omitted points should lie as close to the regression lines as the rest of the data points. The breakpoint for the forward titration (Figure 1A) observed via spectrophotometry was the least apparent. To highlight the distinct breakpoint, even for these data, a single regression line for the complete data set is shown in Figure 1A. The change in slope is 60-70% upon saturation of the protein in these titrations. Typical results obtained via fluorescence titration, monitoring emission of the fluorescein moiety are shown in Figure 2. Note that the data in Figure 2A were used to standardize the concentration of the B4F solution, assuming the protein to be fully competent. As an extension of this project, one might examine other sources of SA to examine whether they are more or less competent than this original sample. The concentration of competent binding sites can also be measured via titration of protein with biotin, monitoring the intrinsic tryptophan fluorescence of the SA (Figure 3).

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r 2010 American Chemical Society and Division of Chemical Education, Inc.

In the Laboratory

Figure 3. Fluorescence titration of biotin into SA (λex = 280 nm, λem = 350 nm.

Discussion Initial executions of this laboratory exercise used a 1-week format with a very prescribed procedure. Instructor observation and student comments indicated a lack of understanding prior to the postexperiment discussion. This feedback gave rise to the current format, which is more guided than directive. Again, based on student feedback, giving the students 1 week to “try out” different dilutions and titration-addition volumes on readily available instruments (benchtop spectrophotometers) gave a more accurate perception of what was going on in the binding assay. Thus, in the second week, students were able to generate data on their own with much less assistance from the instructor. Therefore, we describe this exercise as guided inquiry with stronger guidance in the earlier stages that gradually leaves more of the experimental design and analysis up to the students. Each student group (e.g., 6 groups of two students) shared their data with the rest of the class. With the data from the titration of B4F into SA monitored by fluorescence, students standardized the concentration of the B4F solution. For each data set, students were asked to determine the apparent protein-ligand saturation point. Each group refined the spectrophotometric reverse titration (SA into B4F), so that results could be shared and pooled. The refinement required students to design their experiment to meet particular criteria (i.e., 10-12 data points with at least 3 points before and after the equivalence point). During the next lab period, the students engaged in a discussion where the results from each assay were critiqued (e.g., quality of data, reliability) and began to evaluate the various approaches examined in the project. This discussion typically lasted 45-60 min. A critical analysis of the tools used in this lab included a discussion of the roles that sensitivity, reliability, cost, and throughput play as considerations for choosing a particular assay for a specific application. In this case, one benefit to the SA-B4F system was that the same stock solutions were utilized for a wide working range of concentrations, depending on the instrument used for analysis. In addition to critical analysis of the assay techniques and the examination of protein-ligand binding, we found some other areas of pedagogical significance that have been addressed


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in the design of the project. One of these areas related to the pipetting skills of the students. We worked with students who had already had significant experience using adjustable micropipets, but found that some students were not always careful in their use. Many of the other experiments in our biochemistry curriculum do not depend on highly quantitative pipetting, so the serial pipetting in these titrations quickly highlighted poor technique. Another area related to the ability of the students to design their titration procedure and estimate necessary dilution factors. Although the ranges of concentrations and aliquot sizes were provided, it was up to the students to determine proper dilutions from the concentrated stock solutions. Much of the difficulty seemed to stem from the confluence of skills necessary for this type of experimental design. For example, students must consider how to dilute the analyte and titrant stock solutions, as well as a convenient aliquot size. This required students to use estimation skills, which appear to be relatively unfamiliar to them. This was further exacerbated by the fact that they were being asked to carry out a highly quantitative assay, where the concept of estimation may seem counterintuitive. Because this experiment utilized the same reagents for two distinct assays (i.e., spectrophotometric and fluorescence based), it offered an advantage over a traditional protein-ligand binding experiment as it allowed for investigation of the pros and cons of different assay techniques, emphasizing analytical skills in a biophysical context. Although the assay involved the relatively expensive biotin-4-fluorescein reagent, it was used in very small quantities and allowed for a significant reduction in use of protein (which was rather expensive as well) as compared to the traditional HABA assay. For these reasons, as well as the guided development of the optimized titration protocol, we believe this exercise offers a novel guided-inquiry laboratory project that bridges the biochemistry lab to the physical and analytical laboratory experiences. Acknowledgment We wish to acknowledge the College of Arts and Sciences at John Carroll University for faculty release time, lab supplies, and matching funds. We also wish to thank the Camille and Henry Dreyfus Foundation, Inc., Special Grant Program (SG-01-056) for funding the purchase of the fluorescence spectrometer. Finally, we acknowledge the students in biochemistry and molecular methods laboratories in 2007-2008 for piloting this experiment. Literature Cited


1. Ekins, R. P. J. Chem. Educ. 1999, 76 (6), 769–780. 2. Chial, H. J.; Congdon, R. W.; Splittgerber, A. G. J. Chem. Educ. 1995, 72 (1), 76–79. 3. Marty, A.; Boiret, M.; Deumie, M. J. Chem. Educ. 1986, 63 (4), 365–366. 4. Hansen, D. E.; Tang, D.; Sanborn, J. A.; Marshall, M. D. J. Chem. Educ. 2006, 83 (5), 777–779. 5. Green, N. M. Spectrophotometric Determination of Avidin and Biotin. In Methods in Enzymoogy.; McCormick, D. B., Wright, L. D., Eds.; Academic Press: New York, 1970; Vol. XVIII, pp 418-424. 6. Landman, A. D.; Landman, N. N. J. Chem. Educ. 1976, 53 (9), 591–592.

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7. Prozyme Streptavidin Technical Data. http://www.prozyme.com/ documents/sa10.pdf (accessed Apr 2010). 8. Groman, E. V.; Rothenberg, J. M.; Bayer, E. A.; Wilchek, M. Enzymatic and Radioactive Assays for Biotin, Avidin, an Streptavidin. In Methods in Enzymology; Wilchek, M., Bayer, E. A., Eds.; Academic Press, Inc.: San Diego, CA, 1990; Vol. 184, pp 208-217. 9. Gruber, H. J.; Kada, G.; Marek, M.; Kaiser, K. Biochim. Biophys. Acta 1998, 1381, 203–212. 10. Gruber, H. J.; Marek, M.; Schindler, H.; Kaiser, K. Bioconjugate Chem. 1997, 8, 552–559. 11. Kada, G.; Falk, H.; Gruber, H. J. Biochim. Biophys. Acta 1999, 1427, 33–43. 12. Kim, T. W.; Yoon, H. Y.; Park, J. H.; Kwon, O. H.; Jang, D. J.; Hong, J. I. Org. Lett. 2005, 7 (1), 111–114. 13. Lin, H. J.; Kirsch, J. F. Vitamins and Coenzymes. In Methods in Enzymoogy.; McCormick, D. B.; Wright, L. D., Eds.; Academic Press, Inc.: San Diego, CA, 1979; Vol. 81, pp 287-289. 14. Marek, M.; Kaiser, K.; Gruber, H. J. Bioconjugate Chem. 1997, 8, 560–566.

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15. Mock, D. M.; Horowitz, P. M., Fluorometric Assay for AvidinBiotin Interaction. In Methods in Enzymology; Wilchek, M., Bayer, E. A., Eds.; Academic Press, Inc.: San Diego, CA, 1990; Vol. 184, pp 234-240. 16. Morton, R. C.; Diamandis, E. P. Anal. Chem. 1990, 62 (17), 1841– 1845. 17. Song, X. D.; Swanson, B. I. Anal. Chim. Acta 2001, 442 (1), 79–87. 18. Kada, G.; Kaiser, K.; Falk, H.; Gruber, H. J. Biochim. Biophys. Acta 1999, 1427, 44–48. 19. Waner, M. J.; Mascotti, D. P. J. Biochem. Biophys. Methods 2008, 70, 873–877. 20. Suter, M.; Cazin, J., Jr.; Butler, J. E.; Mock, D. M. J. Immunol. Methods 1988, 113, 83–91.

Supporting Information Available Student handout; instructor notes. This material is available via the Internet at http://pubs.acs.org.


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Complementary Spectroscopic Assays for Investigating Protein

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