In the Laboratory
An Enzyme Kinetics Experiment Using Laccase for General Chemistry
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Yaqi Lin and Patrick M. Lloyd* Department of Physical Sciences, Kingsborough Community College, City University of New York, Brooklyn, NY 11235; *
[email protected] General chemistry laboratory courses often include at least one kinetics experiment. Important concepts covered in kinetics experiments include the effect of reactant concentration, temperature, and the addition of a catalyst on reaction rates. Typically, the effect of a catalyst on reaction rate is a relatively minor part of the kinetics experiment. A common procedure is to add iron(III) ions to catalyze the redox reaction between hydrogen peroxide and iodide ions (1, 2). Another strategy for demonstrating the importance of catalysts is through the use of enzymes. The study of enzymes allows students to synthesize the concepts they have studied in the general chemistry sequence, including kinetics, graphical analysis, spectrophotometry, and, if covered, organic chemistry. Enzymes studied in undergraduate laboratory courses include glucose oxidase, lipase, and catalase (3–5). Enzymes are important in many chemical applications. For example, glucose oxidase, often combined with horseradish peroxidase, can be used to measure concentrations of glucose (3). Measurements of the specific activity of enzymes and the effect of inhibitors on enzyme activity are often covered in biochemistry courses. Issues that have limited the use of enzymes in general chemistry laboratories include the cost of enzymes as well as equipment requirements. A relatively inexpensive and easy to use enzyme, laccase, can be used in general chemistry laboratories as a catalyst in the oxidation of organic compounds. Laccases are a class of oxidoreductase enzymes capable of catalyzing numerous reactions and are found in many species of fungi as well as in certain species of bacteria (6–9). Reactions catalyzed by laccases include the decomposition of lignins in wood and the synthesis of phenolic polymers. Laccases also catalyze the reduction of oxygen to water: O2(g) + 4H+(aq) + 4e− → 2H2O
(l)
Laccases have been studied for use in biofuel cells, toxic waste sites, and as possible replacements for bleaching compounds in the pulp and paper industry (6, 7). Because of their ability to catalyze so many types of reactions there are a number of spectrophotometric assays available for laccases. These involve the oxidation of several common reagents including catechol (1,2-dihydroxybenzene), syringaldazine [4-hydroxyl3,5-dimethoxybenzaldehyde azine (10)], and ABTS [2,2´azinobis-(3-ethylbenzthiazoline-6-sulfonate)]. An advantage in determining the activity of laccases is that they oxidize substrates into easily measured products without the use of secondary reactions. This simplifies measuring the effects of variables like pH and salt concentration. Motivation for the Experiment This experiment is intended as a follow-up to traditional kinetics experiments. In many kinetics experiments students measure first-order or second-order reactions. This experi638
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ment gives students experience with enzyme kinetics and zeroorder reactions. There are several benefits to students performing this experiment. Students are able to measure enzyme activity using a spectrophotometer. This gives students experience in relating absorbance to concentration (Beer–Lambert Law) since the absorbance of the product is proportional to its concentration. Another benefit is that students gain experience with enzymes at an early stage in their chemistry careers. Enzyme kinetics experiments provide a link between kinetics and organic or biochemistry. This experiment also provides an opportunity for students to practice linear regression techniques. Enzyme activity is calculated by graphical methods (fitting data to linear equations). The experiment provides an example of reduction–oxidation chemistry. As mentioned earlier, substrate oxidation is also concomitant with reduction of oxygen to form water. Variables Tested Students test the effect of the substrate on enzyme activity. Two substrates, catechol and syringaldazine, are compared. These substrates have different rates of oxidation that can be measured by spectrophotometric assays. The rate of oxidation of a substrate is related to the size and shape of both the active site of the enzyme and the structure of the substrate. The second variable tested is solution pH. Laccase activity is strongly dependent on solution pH. The pH of maximum activity depends on the species from which the enzyme originates. The enzyme used in this experiment (from T. versicolor, Daiwa Kasei) has a maximum activity near pH 4.5. Students measure the rate of reaction at two pH values, 4.5 and 6.8. The slower rate of reaction at higher pH values promotes the discussion of the importance of the chemical environment to the effectiveness of the enzyme. Laccase activity at lower pH (∼3.0) can also be measured to promote discussion of the importance of hydrogen bonding in protein structure. A third test is the measurement of the effect of adding an inhibitor of the enzyme to the solution. Laccases are inhibited by several chemical species. These include halides, ammonium detergents, some metal ions, and hydroxyglycine. In this experiment sodium chloride is used as the inhibitor. Millimolar concentrations of chloride can reduce the rate of reaction for laccase. Data analysis for this experiment involves the calculation of laccase activity. The measured absorbance for syringaldazine (molar extinction coefficient is 65,000 M᎑1 cm᎑1 at 525 nm) is used to calculate the number of moles of syringaldazine turned over per second. This allows students to determine the approximate number of moles of substrate involved in the reaction.
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In the Laboratory
Procedure
1.6 ⴚ3
y = (8.0 ⫻ 10 R 2 = 1.0
Absorbance
1.4 1.2
ⴚ2
)x + 3.3 ⫻ 10
1.0 0.8 0.6
y = (2.1⫻10ⴚ4)x + 6.2 ⫻ 10ⴚ2 R 2 = 0.99
0.4 0.2 0.0 60
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Time / s
1.6
y = (8.0 ⫻ 10 R 2 = 1.0
Absorbance
1.4 1.2
ⴚ3
)x + 3.3 ⫻ 10
ⴚ2
1.0 0.8 0.6
y = (2.9 ⫻ 10ⴚ4)x + 6.4 ⫻ 10ⴚ2 R 2 = 1.0
0.4 0.2 0.0 60
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Time / s Figure 2. Syringaldazine assay and the effect of solution pH on laccase activity: (䊊) pH = 4.5 and (䊏) pH = 6.8.
1.6
y = (8.0 ⫻ 10 R 2 = 1.0
1.4 1.2
ⴚ3
)x + 3.3 ⫻ 10
ⴚ2
1.0 0.8 0.6
y = (1.2 ⫻ 10ⴚ3)x + 1.8 ⫻ 10ⴚ2 R 2 = 0.99
0.4
Hazards
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Figure 1. Laccase activity and the effect of the substrate at pH 4.5. Measurements for the syringaldazine assay ( 䊊) were made at 525 nm. For the catechol assay (䊏) measurements were made at 410 nm.
Absorbance
Students work in groups of two or three using a spectrophotometer with digital display (Spectronic 301), a mechanical time counter, graduated pipets, and solutions. Each group is given solutions containing the enzyme (0.32 mg兾mL in 50 mM pH 4.5 sodium acetate buffer) and separate solutions containing 5 × 10᎑4 M syringaldazine (Aldrich) in ethanol and catechol (Aldrich) in 50 mM pH 4.5 sodium acetate buffer. Students perform the experiment using a digital display spectrophotometer. These instruments are becoming more common in general chemistry laboratories. Instruments with analog displays can also be used. However, digital displays make data acquisition easier for students. Stopwatches can be used for time keeping. For assays using syringaldazine students perform a blank measurement using the buffer and the substrate solution. Students then add the enzyme solution to the mixture and start the timer. For the catechol assay, the substrate is pre-mixed with the buffer. The substrate–buffer mixture is used as a blank and then the enzyme solution is added. One group member announces the time intervals (typically every 20 seconds) and another group member measures the absorbance value and writes the absorbance value into a laboratory notebook. This procedure is repeated two more times so that there are three assays for each solution. In all, the experiment takes about two hours for the average group of students to complete. Advanced students can complete the procedure in an hour and a half. A strategy to save time and promote teamwork is to break the class into several groups. Each group is responsible for measuring two of the four solutions. In this procedure the entire class shares data and each solution is assayed by two groups for confirmation of results. Another method for saving time is to substitute droppers for graduated pipets. If two laboratory periods are available then there are additional studies that can be made, for example, the effect of additional pH values on enzyme activity, the effect of inhibitor concentration on enzyme activity, measurements of absorption spectra of the substrate products, and visible absorption spectra of the products.
0.2
Catechol is toxic and should be washed from skin on contact. Syringaldazine is an irritant and should be washed from skin on contact. Laccase is an oxidoreductase enzyme and should be washed from skin on contact. Results and Discussion Results from student measurements are shown in Figures 1–3. Numerical results are shown in Table 1. Measurements with syringaldazine as substrate were performed at 525 nm. Measurements with catechol as the substrate were performed at 410 nm. Syringaldazine solutions, when oxidized, change from a pale yellow to a bright purple color. Catechol solutions change from colorless to a dark greenish-gray color when oxidized. The slope of the absorbance change over time determines enzyme activity. Typical data are shown for the syringaldazine and catechol assays on Figure 1. The greatest activity is found with syringaldazine as substrate at pH 4.5. www.JCE.DivCHED.org
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Time / s Figure 3. Syringaldazine assay and the effect of chloride inhibitor on laccase activity at pH 4.5: (䊊) 0 mM chloride and (䊏) 6.2 mM chloride.
Table 1. Student Results for Laccase Activity Solution
pH
Activity/(U/mL)a
Syringaldazine
4.5
18.8 ± 4.63
Catechol
4.5
1.04 ± 0.51
Syringaldazine
6.8
0.90 ± 0.50
Syringaldazine–chlorideb
4.5
7.87 ± 3.14
a Data for two laboratory sections (12 groups) are aggregated. One activity unit is defined as a change of 0.001 absorbance units per minute at 525 nm for syringaldazine and 410 nm for catechol. bThe chloride concentration is 6.2 mM.
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In the Laboratory
Laccase activity using syringaldazine as substrate is about twenty times greater than with catechol. Maximum activity of the laccase used in this experiment occurs at pH 4.5 to 5.0. Measurements at pH 6.8 compared to those at pH 4.5 show that the activity is reduced with an increase in pH (Figure 2). An explanation for the pH-dependent reduction in laccase activity is that the hydroxide ion acts as an inhibitor. Another possibility is that hydrogen bonding within the enzyme may be affected with a change in pH, resulting in decreased activity. There are many known inhibitors of laccase. In this experiment chloride was tested by adding a small volume of 7.5 mM sodium chloride solution (diluted to 6.2 mM) to a pH 4.5 buffered solution (Figure 3). The addition of chloride reduced the activity by a factor of about sixty percent relative to the solution without chloride. Higher concentrations of chloride result in even greater loss of enzyme activity (results not shown). Summary The use of enzymes in general chemistry laboratories presents an opportunity to discuss several important topics. Laccase is a useful enzyme for demonstrating these topics. The effect of pH, chemical environment, and shape of the substrate can all be easily tested using laccase. This experiment shows that students can measure these variables and gain a better understanding of the importance of kinetics and catalysis and also gain an appreciation for spectrophotometry and graphical methods. In many institutions the majority of freshmen chemistry students are biology or premed majors. Student reaction to the laccase experiment has in-
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cluded fascination with the clear connection between biology and chemistry and relevance of chemistry to biological concepts. Acknowledgments The authors thank Chin Yee Lum and Pavel Sigel for their work on this project. W
Supplemental Material
Instructions for the students and notes for the instructor are available in this issue of JCE Online. Literature Cited 1. Hansen, J. C. J. Chem. Educ. 1996, 73, 728. 2. McAlpine, R. K. J. Chem. Educ. 1945, 22, 387. 3. Vasilarou, A. M. G.; Georgiou, C. A. J. Chem. Educ. 2000, 77, 1327. 4. Farley, K. A.; Jones, M. A. Chem. Educ. 1989, 66, 524. 5. Kimbrough, D. R.; Magoun, M. A.; Langfur, M. J. Chem. Educ. 1997, 74, 210. 6. Gianfreda, L.; Xu, F.; Bollag, J. M. Biorem. J. 1999, 3, 1. 7. Li, K.; Helm, R. F.; Eriksson, K. E. Biotechnol. Appl. Biochem. 1998, 27, 239. 8. Ghindilis, A. Biochem. Soc. Trans. 2000, 28, 84. 9. Yaropolov, A. I.; Skorobogatko, O. V.; Vartanov, S. S.; Varfolomeyev, S. D. Appl. Biochem. And Biotech. 1994, 49, 257. 10. Leonowica, A.; Grzywnowicz, K. Enzyme Microb. Technol. 1981, 3, 55.
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