In the Classroom
Tested Demonstrations
Factors Affecting Reaction Kinetics of Glucose Oxidase submitted by:
checked by:
Kristin A. Johnson† Department of Chemistry, University of Wisconsin, Madison, WI 53706;
[email protected] Beth A. Kroa and Tony Yourey
Department of Chemistry, Kutztown University, Kutztown, PA 19530
There has been an increased emphasis in recent years on teaching the basic principles of biochemistry in undergraduate general chemistry classes. Although a number of biochemistry laboratory experiments have been adapted to the general chemistry curriculum (1–4), there is a notable absence of demonstrations on this material. This paper describes a demonstration based on a biochemical kinetics laboratory experiment, using the enzyme glucose oxidase (4), and includes a method that employs horseradish peroxidase from fresh horseradish root. The rate of the glucose oxidase reaction varies with the enzyme concentration, substrate concentration, substrate used in the reaction, and the reaction temperature. Glucose oxidase catalyzes the oxidation of β-D-glucose to δ-gluconolactone through a two-step reaction (Scheme I). H HO HO
CH2 H O
H H H
O2
H2O2
OH
E•FAD
E•FADH2
OH OH
H HO HO
OH CH2 H O H
OH O
H
Scheme I
In the first part of the reaction, β-D-glucose is oxidized to δgluconolactone by the glucose oxidase cofactor, flavin adenine dinucleotide (FAD), which in turn is reduced to FADH2. In the second step, oxygen is reduced to hydrogen peroxide, and FADH2 is reoxidized to FAD (5). Because both β-D-glucose and δ-gluconolactone are colorless in solution, the rate is determined by monitoring the release of hydrogen peroxide, using horseradish peroxidase and a chromogenic substrate. The hydrogen peroxide produced by the glucose oxidase reaction is enzymatically reduced to water by horseradish peroxidase. †
Mailing address: 6816 E. 65th St., Indianapolis, IN 46220.
The overall reaction is H2O2 + AH2 → A + 2H2O
where AH2 is the electron donor. In this experiment, 2,2′azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) serves as the electron donor (4, 6 ). It is virtually colorless in its reduced form, but forms a bright blue-green color when oxidized. Horseradish peroxidase and ABTS are in excess, so the rate of color formation is dependent on the hydrogen peroxide produced in the glucose oxidase reaction. Preparation Prepare 3.0 L of 0.1 M potassium phosphate buffer, pH 7.0, by combining 186 mL of 1 M K2HPO4, 114 mL of 1 M KH2PO4, and 2700 mL of distilled water. Glucose oxidase (G 6125), horseradish peroxidase (P 8125), ABTS, and 2deoxyglucose were purchased from Sigma Chemical Company, St. Louis, MO. Prepare 60 mL of glucose oxidase solution, 2 units per milliliter (U/mL), in phosphate buffer. Keep on ice until ready for use. Prepare 15 mL of a solution of 50 mM ABTS and 25 U/mL of horseradish peroxidase (HRP) in 0.1 M potassium phosphate buffer. It may be necessary to warm the solution gently to dissolve the ABTS. Alternatively, horseradish extract can be used as a substitute for pure horseradish peroxidase. Purée 25 g of horseradish root with 50 mL of 0.1 M potassium phosphate buffer. Remove the solid particles from the mixture by filtering through cheese cloth or a kitchen strainer. For best results, all solutions should be used within two days or less of their preparation. Prepare 1 M stock solutions of glucose, maltose, fructose, and 2deoxyglucose. The quantity and composition of the buffer, glucose oxidase, and substrate samples needed for the demonstration are summarized in Table 1. Arrange thirteen 300-mL tall-form beakers on the bench top. Pour 200 mL of potassium phos-
Table 1. Buffer, Glucose Oxidase, and Substrate Samples Needed for the Demonstration Glucose Oxidase, in Test Tubes on Ice
Buffer, in 300-mL Tall-Form Beakers No. Contents
No. Contents
No. Contents
200 mL phosphate buffer and 1 mL ABTS/HRP at room temp
10
5 mL, 2 U/mL
8
10 mL, 1 M glucose
1
5 mL, 2 U/mL at 80°C
1
10 mL, 0.5 M glucose
200 mL phosphate buffer and 1 mL ABTS/HRP at 80 °C
1
5 mL, 0.2 U/mL
1
10 mL, 0.05 M glucose
1
5 mL, 0.02 U/mL
1
10 mL, 1 M maltose
1
10 mL, 1 M fructose
1
10 mL, 1 M 2-deoxyglucose
1
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Substrate, in Test Tubes
11 1
200 mL phosphate buffer and 1 mL ABTS/HRP at 4 °C
(1)
Journal of Chemical Education • Vol. 79 No. 1 January 2002 • JChemEd.chem.wisc.edu
In the Classroom
phate buffer and 1 mL of ABTS/HRP solution into each beaker. If the horseradish extract is used, add 1 mL of 50 mM ABTS solution and 1 mL of the horseradish extract to each beaker. The enzymatic reaction will be carried out in these beakers. The color of the product is more visible if the beakers are placed in front of a white background. Chill one beaker containing the buffer and ABTS/HRP solution on ice to 4 °C, and heat another to approximately 80 °C. Place 10 test tubes on ice. Pour 5 mL of the 2 U/mL glucose oxidase stock solution into each test tube. Heat a beaker of water to approximately 80 °C. Pour 5 mL of the 2 U/mL glucose oxidase solution into a test tube and place the test tube into the beaker of hot water for at least 5 minutes to heat-inactivate the enzyme. Dilute a portion of the glucose oxidase stock solution 1:10 with buffer, and put 5 mL of the diluted solution into a test tube on ice. Dilute the glucose oxidase stock solution 1:100 with buffer and put 5 mL of that solution into a test tube on ice. Label all the test tubes. Pour 10 mL of the 1 M glucose into each of 8 test tubes. Prepare 10 mL 0.5 M glucose and pour into a test tube. Prepare 10 mL of 0.05 M glucose, and pour that solution into a test tube. Pour 10 mL of 1 M maltose, 10 mL of 1 M fructose, and 10 mL of 1 M 2-deoxyglucose into three different test tubes. Label all the tubes appropriately. Demonstration
Changes in Reaction Kinetics with Enzyme Concentration To test how the enzyme concentration affects the reaction kinetics, pour the contents of 3 test tubes containing 10 mL of 1 M glucose into 3 different beakers containing buffer and ABTS/HRP at room temperature. Add 5 mL of 2 U/mL glucose oxidase to one beaker, 5 mL of 0.2 U/mL glucose oxidase to the second beaker, and 5 mL of 0.02 U/mL glucose oxidase to the third beaker. The concentrations of glucose oxidase and glucose in each beaker for this series of reactions are listed in Table 2. Stir gently. Note the differences in rate of color appearance. Changes in Reaction Kinetics with Substrate Concentration To test how the rate of the reaction varies with substrate concentration, pour the contents of 3 test tubes containing 5 mL of 2 U/mL glucose oxidase into 3 beakers containing buffer and ABTS/HRP at room temperature. Add 10 mL of 1 M glucose to one beaker, 10 mL of 0.5 M glucose to the second beaker, and 10 mL of 0.05 M glucose to the third beaker. The concentrations of glucose oxidase and glucose in each beaker for this part of the demonstration are listed in Table 3. Stir gently. Note the differences in rate of color appearance. Substrate Specificity of Glucose Oxidase To show how the rate of the glucose oxidase reaction changes depending on the substrate of the reaction, pour the contents of 4 test tubes containing 5 mL of 2 U/mL glucose oxidase into 4 beakers containing buffer and ABTS/HRP at room temperature. Add 10 mL of 1 M glucose to one beaker, 10 mL of 1 M 2-deoxyglucose to the second beaker, 10 mL of 1 M fructose to the third beaker, and 10 mL of 1 M maltose to the fourth beaker. The final concentration of substrate is the
Table 2. Concentrations in Each Beaker when Enzyme Concentration Is Varied (25 ° C) Beaker
Glucose Oxidase
Glucose Substrate
Amount Added
Final Concn
Amount Added Final Concn
1
5 mL, 2 U/mL
0.05 U/mL
10 mL, 1 M
0.046 M
2
5 mL, 0.2 U/mL
0.005 U/mL
10 mL, 1 M
0.046 M
3
5 mL, 0.02 U/mL
0.0005 U/mL
10 mL, 1 M
0.046 M
Table 3. Concentrations in Each Beaker when Substrate Concentration Is Varied (25 ° C) Beaker
Glucose Oxidase
Glucose Substrate
Amount Added
Final Concn
Amount Added
Final Concn
4
5 mL, 2 U/mL
0.05 U/mL
10 mL 1 M
0.046 M
5
5 mL, 2 U/mL
0.05 U/mL
10 mL 0.5 M
0.023 M
6
5 mL, 2 U/mL
0.05 U/mL
10 mL 0.05 M
0.0023 M
same in all beakers. Stir gently. Note the differences in rate of color appearance.
Temperature Dependence of Reaction Rate To test how the rate of the reaction changes with reaction temperature, add the contents of 3 test tubes containing 10 mL of 1 M glucose to three beakers containing buffer and ABTS/HRP. One beaker should be at 4 °C, the second at room temperature, and the third at 80 °C. Add 5 mL of 2 U/mL glucose oxidase to the beakers at 4 °C and room temperature. Add the heat-treated glucose oxidase to the beaker at 80 °C. In this series, the concentrations of enzyme and substrate are the same in all beakers. Stir gently. Observe the rate of color formation at each temperature. Disposal All solutions can be poured down the drain. Discussion Glucose oxidase catalyzes the formation of δ gluconolactone from β-D-glucose. The rate of the enzymatic reaction can be varied by changing the enzyme concentration, substrate concentration, substrate used, and reaction temperature. As shown in the first part of the demonstration, the rate at which the colored oxidized ABTS substrate appears is dependent on the concentration of enzyme used in the reaction. The fastest reaction had 0.05 U/mL of the enzyme, whereas the slowest had only 0.0005 U/mL. At constant enzyme concentration, the velocity of the enzymatic reaction varies with substrate concentration. When a very dilute solution of glucose was used, the rate was significantly slower. However, there was not a significant difference between the reaction rates when the concentration of glucose was 0.046 M and 0.023 M. The amount of substrate is saturating at these concentrations, and increasing the substrate concentration does not increase the rate of the reaction. One of the distinct characteristics of glucose oxidase is its extremely high substrate specificity (7). Any alteration in the molecular structure of the substrate from β-D-glucose can enormously reduce the rate of oxidation. The only variant of
JChemEd.chem.wisc.edu • Vol. 79 No. 1 January 2002 • Journal of Chemical Education
75
In the Classroom
glucose that is oxidized at an appreciable rate is 2-deoxy-Dglucose, which is fourfold slower than glucose itself. The high degree of specificity of glucose oxidase makes it useful for the detection and estimation of glucose in biological material. It is widely used in combination with other enzymes and chemicals in blood glucose meters commonly used by diabetics. The last part of the demonstration shows the effect of temperature on enzymatic reactions. There is a significant increase in the rate of the reaction from 4 °C to room temperature. Although the rate of most enzymatic reactions increases with temperature, if the temperature gets too high the reaction will not proceed. At elevated temperatures, the tertiary structure of the protein is disrupted and the enzyme essentially unravels and is not active.
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Literature Cited 1. Hamilton, T. M.; Dobie-Galuska, A. A.; Wietstock, S. M. J. Chem. Educ. 1999, 76, 642. 2. Nordell, K. J.; Jackelen, A.-M. L.; Condren, S. M.; Lisensky, G. C.; Ellis, A. B. J. Chem. Educ. 1999, 76, 400A. 3. Hershlag, N.; Hurley, I.; Woodward, J. J. Chem. Educ. 1998, 75, 1270. 4. Bateman, R. C.; Evans, J. A. J. Chem. Educ. 1995, 72, A240. 5. Wong, D. W. S. Food Enzymes: Structure and Mechanism; Chapman & Hall: New York, 1995. 6. Childs, R. E.; Bardsley, W. G. Biochem. J. 1975, 145, 93. 7. Dixon, M.; Webb, E. Enzymes; Academic: New York, 1979; pp 243–244, 479–485.
Journal of Chemical Education • Vol. 79 No. 1 January 2002 • JChemEd.chem.wisc.edu
