In the Laboratory
The o -Phenylenediamine-Horseradish Peroxidase System: W Enzyme Kinetics in the General Chemistry Laboratory T. M. Hamilton,*† A. A. Dobie-Galuska, and S. M. Wietstock Department of Chemistry, Indiana University, Bloomington, IN 47405; *
[email protected] Kinetics experiments are an essential component of undergraduate chemistry laboratory courses. They introduce students to the important dimension of time in chemical reactions and are especially beneficial in that they also introduce students to biochemistry. With these goals in mind, we developed an enzyme kinetics laboratory experiment that can be easily incorporated into an undergraduate chemistry program. The purpose of the experiment is to measure the kinetic parameters in the oxidative coupling of o-phenylenediamine (OPD) to 2,3-diaminophenazine (DAP) (1), a reaction that is catalyzed by the enzyme horseradish peroxidase (HRP). Horseradish peroxidase, a protein with a mass of 34 kDa, is found in the root of the horseradish plant, where its natural function is the oxidation of aromatic substrate molecules (2). The oxidative coupling of OPD to DAP is widely used as a catalymetric reaction for the assay of many transition metal ions (3). OPD is also an effective chromogenic substrate for HRP-mediated enzyme-linked immunosorbent assays (4, 5). The intensity of the colored product is measured by a spectrophotometer at a wavelength of 450 nm: NH2 2
N
NH2
N
NH2
HRP/H2O2
NH2
o-phenylenediamine (OPD)
2,3-diaminophenazine (DAP: orange-brown)
Students follow the rate of the reaction by monitoring the change in absorbance intensity with time. Several kinetic runs are performed with varying OPD concentrations and a fixed HRP concentration. The kinetics parameters (maximum reaction velocity, Michaelis constant, and maximum turnover number) are determined by straight-line plots of variations of the Michaelis–Menten equation, such as the Lineweaver– Burk or Eadie–Hoffstee plot (6–10). Experimental Procedure
Alternatively, conventional UV–vis spectrophotometers linked to strip-chart recorders by means of an antilog amplifier could be used for measuring the kinetic runs. The experiment may as well be performed with a simple spectrophotometer, such as the Spectronic 20 (Milton Roy), with manual recording of the data.
Stock Solutions 1. 0.05 M 2-[N-morpholino]ethane sulfonic acid (MES) hemisodium salt (Sigma) buffer, pH 6.0. 2. 0.0022 M o-phenylenediamine (OPD) dihydrochloride (Sigma) in 0.05 M MES buffer, pH 6.0. Add 1.6 µ L of hydrogen peroxide (30%) per milliliter of OPD solution immediately prior to use. 3. Horseradish peroxidase (HRP) (Type I, Sigma): 3.5 × 10{8 M in 0.05 M MES buffer, pH 6.0.
WARNING: OPD is a suspected carcinogen. Latex gloves should be worn in preparing and using the OPD solutions. All solutions were allowed to warm to room temperature (25 °C) before use. The HRP solution can be stored at 4 °C for two days. OPD photodecomposes in aqueous solution and turns yellow; consequently the OPD solution must be made fresh each day.
Experimental Protocol The experiment was designed to be performed in one laboratory period (2–3 hours) with students working in pairs. The kinetic runs were performed by using varying volumes of the buffer (solution 1) and OPD (solution 2) mixed with 1 mL of the HRP (solution 3) in a total volume of 6 mL. After mixing, a portion of the solution was transferred to a 4-mL polystyrene cuvette and placed in the colorimeter sample holder. One can determine the extinction coefficient of the product, 2,3-diaminophenazine (DAP), since it is commercially available as a custom-order compound. However, owing to its high cost, its purchase is not an option for some chemistry
Equipment Colorimeters (Vernier Software, Portland, OR) interfaced to personal computers via the LabVIEW programming language (National Instruments, Austin, TX) were used in the experiments (the Virtual Instrument is available as supplementary material via JCE Online).W One is able to select one of three wavelengths with the colorimeter: 470, 565, or 635 nm. We chose 470 nm, the one closest to the recommended wavelength of 450 nm. The transmittance of the DAP product was measured at 1-s intervals and converted to absorbance in the LabVIEW program. Absorbance versus time was plotted in real time on the personal computer screen (see Fig. 1). † Present address: Department of Chemistry, Adrian College, Adrian, MI 49221.
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Figure 1. Personal computer screen output from LabVIEW Enzyme Kinetics VI.
Journal of Chemical Education • Vol. 76 No. 5 May 1999 • JChemEd.chem.wisc.edu
In the Laboratory
programs. We determined the extinction coefficient for DAP by making the assumption that all of the substrate (OPD) had been converted into product (DAP) at the point when there is no further change in absorbance (Amax, Fig. 2). Since two OPD molecules are required to produce one DAP molecule (1), the DAP concentration at the maximum value of absorbance is equal to one-half the original OPD concentration. A Beer’s law plot was constructed using the Amax and corresponding [DAP] values from several runs and the extinction coefficient derived from this plot was used to calculate the initial reaction velocities. Results and Discussion
Kinetics Parameters The experiment has been performed in three-hour laboratory periods by 674 first-year undergraduate students (working in pairs) for the past three semesters. Students began acquiring data 20 s after mixing the substrate and enzyme solutions. The initial reaction velocities were determined from the slope of the absorbance-vs-time plots for data points between 20 and 35 s. The mixing time and number of data points used to calculate the slope can be varied; however, care must be taken to assure that the points obtained are within the linear portion of the absorbance–time plot. The initial reaction velocities were calculated from the following formula (11): vinitial = slopeinitial/εOPD At the end of 35 s, the reaction is 30–35% complete. Ideally, the initial velocity would be better measured when the reaction is but a few percent complete (6 ); however, reproducible results were obtained with the above procedure. Students constructed Lineweaver–Burk plots (1/v vs 1/[OPD]) and determined the kinetics parameters. A sample plot is shown in Figure 3. The following ranges of values for the kinetic parameters were obtained: vmax = 6.5 ± 1.5 × 10{3 mM s{1, KM = 0.60 ± 0.20 mM, k0 (turnover number) = 1.15 ± 0.15 × 103 s{1. The solutions had substrate values bracketing KM, that is, [OPD] = 0.1 to 1.0 mM. Students also calculated the activity using a single OPD concentration ([OPD] = 0.74 mM); values were in the range of 1.12 ± 0.19 × 103 µmol DAP min{1 mg{1 HRP. Follow-up questions addressed topics such as (i) the significance of vmax, KM, and the turnover number; (ii) the origin of the absorbance saturation after extended
time periods; and (iii) the effect of HRP concentration on the amount of DAP formed in the reaction. Variations A variation that would be more suitable for an elementary chemistry laboratory would be a set of runs in which the student varies the HRP concentration while holding the OPD concentration constant. The absorbance is read after a specific time interval, for example 50 s. The corresponding DAP concentration is plotted vs the HRP concentration to demonstrate the linear relationship between the amount of product formed and the enzyme concentration. We used an OPD concentration of 0.74 mM and HRP concentrations of 5.9 × 10{10 to 5.9 × 10 {9 M (0.020 to 0.20 mg/L) and observed slopes near 1.0 mM DAP mg {1 L{1 HRP. For honors or upper-level courses, the effect of inhibitors on the kinetics can be explored. A number of inorganic and organic substances inhibit the OPD–HRP system (12). The following ions cause 50% inhibition in the millimolar concentration range (the corresponding concentration is given in parentheses): Cu 2+ (6.46 × 10{4 M), CN{ (6.76 × 10{4 M), sulfite (1.06 × 10-3 M), cystine (3.84 × 10-3 M), Cd2+ (4.17 × 10{3 M), Ni2+ (5.25 × 10{3 M). The type of inhibition (e.g., competitive, noncompetitive, mixed) can be ascertained if one changes the inhibitor concentration and observes changes in the linear portions of the plots originating from the application of the Michaelis–Menten equation. The students are given the inhibitor concentrations and are asked to evaluate the inhibition constant, K i. Follow-up questions focus on the mechanistic differences between the various types of inhibition, discussion of how the ion might bind to the heme active site on HRP (in the case of competitive inhibition) or how the ion prevents the formation of the OPD–HRP complex (in the case of noncompetitive inhibition), and treatment of errors in the straight-line plots. Examples of questions that might be posed are as follows: 1. Explain the mechanism by which a competitive inhibitor decreases the amount of product formed per unit time in an enzyme-catalyzed reaction. 2. What type of interaction does a noncompetitive inhibitor have with the enzyme? 3. Did the inhibitor that you used manifest competitive, noncompetitive, or mixed inhibition? Would you expect the observed type of inhibition based upon the structure of the inhibitor and fact that HRP has a heme active site?
Amax
1/v / (s L/mmol)
tia
700
ini
Absorbance
l ra te
800
600 500 400 300 200 100 0
Time / s Figure 2. Plot of absorbance vs time in a kinetic run of the OPD– HRP system.
-4
-2
0
2
4
6
8
10
1/[OPD] / (L/mmol) Figure 3. A sample Lineweaver–Burk plot ([HRP] = 5.9 × 10{ 9 M).
JChemEd.chem.wisc.edu • Vol. 76 No. 5 May 1999 • Journal of Chemical Education
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In the Laboratory
Acknowledgments
Literature Cited
Funding for the hardware and software used in this project came in part from the following three grants to Adam Allerhand: Award DUE-9650303 from the National Science Foundation, Award No. SG-96-031 from the Camille and Henry Dreyfus Foundation, and a grant from the Office of Information Technologies of Indiana University. We appreciate the work of Chris Stavitzke, participant in the 1997 National Science Foundation–supported Exploration of Careers in Science Program at Indiana University, in testing the peroxidase inhibitors. We would also like to thank the Indiana University students who helped test the experiment, particularly Todd Eads and Tim Von Fange. Special thanks to M. Jackson and T. Hacker for preparing the chemicals and equipment for the students and to D. Peters, Professor of Chemistry, for the use of his laboratory. Note
1. Tarcha, P. J.; Chu, V. P.; Whittern, D. Anal. Biochem. 1987, 165, 230–233. 2. Friedrich, J. Methods in Enzymology: Biochemical Spectroscopy; Sauer, K., Ed.; Academic: New York, 1995; Vol. 246, p 252. 3. Mekler, V. M.; Bystryak, S. M. J. Photochem. Photobiol. A: Chem. 1992, 65, 391–397. 4. Mekler, V. M.; Bystryak, S. M. Anal. Chim. Acta 1992, 264, 359– 363. 5. Bovaird, J. H.; Ngo, T. T; Lenhoff, H. M. Clin. Chem. 1982, 28, 2423–2426. 6. Cornish-Bowden, A. Fundamentals of Enzyme Kinetics, Rev. ed.; Portland: London, 1995; pp 30–37, 56–57. 7. Ault, A. J. Chem. Educ. 1974, 51, 381. 8. Moe, O.; Cornelius, R. J. Chem. Educ. 1988, 65, 137. 9. Smith, W. G. J. Chem. Educ. 1992, 69, 981. 10. Bateman, R. C. Jr.; Evans, J. A. J. Chem. Educ. 1995, 72, A240. 11. Anderson, J.; Byrne, T; Woelfel, K. J.; Meany, J. E.; Spyridis, G. T.; Pocker, Y. J. Chem. Educ. 1994, 71, 715. 12. Guilbault, G. G.; Brignac, P. Jr.; Zimmer, M. Anal. Chem. 1968, 40, 191.
W Supplementary materials for this article are available on JCE Online at http://JChemEd.chem.wisc.edu/Journal/Issues/1999/May/ abs642.html.
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Journal of Chemical Education • Vol. 76 No. 5 May 1999 • JChemEd.chem.wisc.edu
