In the Classroom
Tested Demonstrations
A Simple Method for Demonstrating Enzyme Kinetics Using Catalase from Beef Liver Extract submitted by:
Kristin A. Johnson Department of Chemistry, University of Wisconsin–Madison, Madison, WI 53706;
[email protected] checked by:
William C. Deese Department of Chemistry, Louisiana Tech University, Ruston, LA 71272
Laboratory investigations of enzyme kinetics are common in undergraduate biochemistry and even general chemistry classes (1–6 ). In most cases, the enzymatic reaction produces a colored product, the intensity of which is measured using a spectrophotometer. Unfortunately, these types of reactions have limited value as demonstrations, especially if data collection is desired. Watching a spectrophotometer has limited visual appeal and the class does not see the reaction itself. This paper describes a simple visual method of demonstrating enzyme kinetics using beef liver catalase. Catalase is a heme-containing enzyme that is ubiquitous in aerobic organisms. It catalyzes the decomposition of hydrogen peroxide to water and oxygen: 2H2O2 → O2 + 2H2O(ᐉ) In the demonstration, a catalase solution is obtained by homogenizing fresh beef liver in a phosphate buffer. Filter paper is saturated with beef liver extract and placed into a solution of hydrogen peroxide. Oxygen forms on the filter paper, and the filter paper rises to the top of the beaker. Catalase activity is measured by timing the rise of the enzymesoaked filter paper to the top of beakers containing different concentrations of hydrogen peroxide (6 ). The data are plotted as a Lineweaver–Burk double-reciprocal plot, and the Km and Vmax for the reaction are calculated. Materials 4 g fresh or frozen beef liver 500 mL ice-cold 0.1 M potassium phosphate buffer, pH 7.0 (To prepare, mix 31 mL of 1 M K2HPO4, 19 mL of 1 M KH2PO4, and 450 mL of distilled water.) blender 500 mL 2% hydrogen peroxide solution (To prepare, dilute 34 mL of 30% H2O2 to 500 mL with water. CAUTION: 30% hydrogen peroxide is a very strong oxidizing agent. Always wear gloves when handling.) 500 mL 1% hydrogen peroxide solution (To prepare, dilute 17 mL of 30% H2O2 to 500 mL with water.) 500 mL 0.5 % hydrogen peroxide solution (To prepare, dilute 83 mL 3% H2O2 to 500 mL with water)
500 mL distilled water six 600-mL beakers 1 box of 42.5-mm diameter hardened Whatman’s No. 50 filter paper one 125 × 65-mm crystallizing dish stopwatch forceps, long enough to reach to bottom of 600-mL beaker ice and ice bucket paper towels computer with graphing program such as Microsoft Excel or Cricket Graph installed
Preparation Fresh beef liver is available at many local grocery stores. If fresh liver is not available, adequate results can be obtained with frozen beef liver. Homogenize 4 g of the liver in a blender with 250 mL of ice-cold 0.1 M potassium phosphate buffer pH 7.0. Freeze the remaining liver at ᎑20 °C for subsequent use. Dilute 25 mL of the homogenized liver solution 1:10 with ice-cold 0.1 M potassium phosphate buffer pH 7.0 and keep on ice in a 125 × 65-mm crystallizing dish. Place a few pieces of 42.5-mm diameter hardened Whatman’s No. 50 filter paper in the liver extract. More can be added later, depending on how many trials are performed. Prepare 500 mL each of 2%, 1%, 0.5%, 0.3%, and 0.2% hydrogen peroxide solutions in 600-mL beakers and place 500 mL of water in another 600-mL beaker. Since the catalase concentration in the extract can vary between preparations, a trial run of the demonstration at the highest and lowest concentrations of hydrogen peroxide will ensure proper presentation. There should be approximately a threefold difference in the time it takes for the filter paper to rise to the top of the beaker between the highest and lowest hydrogen peroxide concentration. If the time required for the filter paper to rise to the top of the beaker at the lowest hydrogen peroxide concentration is longer than 40 seconds, the catalase is too dilute. Add more concentrated liver extract or dilute the existing extract appropriately to achieve the proper ratio.
500 mL 0.3% hydrogen peroxide solution (To prepare, dilute 50 mL of 3% H2O2 to 500 mL with water.)
Presentation
500 mL 0.2% hydrogen peroxide solution (To prepare, dilute 34 mL of 3% H2O2 to 500 mL with water.)
As an introduction to this demonstration, take one piece of filter paper from the beef liver extract and blot it gently on
JChemEd.chem.wisc.edu • Vol. 77 No. 11 November 2000 • Journal of Chemical Education
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In the Classroom
paper towels to remove excess extract. Using forceps, place the filter paper flat on the bottom of the beaker of water. The filter paper will not rise to the top. Repeat the process with another piece of extract soaked filter paper in the 2% hydrogen peroxide solution. Oxygen bubbles will form noticeably around the filter paper and it will quickly rise to the top of the beaker. Repeat the process with each hydrogen peroxide solution. Time how long it takes the filter paper to rise to the top of the beaker at each concentration. Stop the timer when the center of the piece of filter paper reaches the top of the hydrogen peroxide solution. The filter paper will often turn sideways while rising to the top, so measuring the time it takes for the center of the piece of paper to reach the top will ensure consistency throughout the data set. Several trials should be performed for each hydrogen peroxide concentration. To speed up the demonstration, several time intervals can be measured at the same time. Gather volunteers, and assign one person to each concentration of hydrogen peroxide. Simultaneously add a piece of filter paper to each of the beakers and record the time it takes for the filter paper to rise to the top of the hydrogen peroxide solution. Since the actual amount of hydrogen peroxide converted to oxygen and water in each trial is minimal, it can be assumed that the hydrogen peroxide concentration in each beaker remains constant over the course of the demonstration. Occasionally, the filter paper will stick to the side of the beaker. Those data points should be discarded. The number of discarded time points can be minimized by not allowing the filter paper to soak in the extract for too long. A typical data set is shown in Table 1. It includes the average of times from at least three trials. The velocity at each substrate concentration is calculated by taking the reciprocal of the average time for each concentration. Reciprocals of the substrate concentration and the velocity were also calculated, since these data will be plotted on a Lineweaver–Burk doublereciprocal plot. The data are plotted using a computer graphing program such as Microsoft Excel or Cricket Graph (Fig. 1). Use the computer program to generate a best linear fit line between the points and an equation for the line. The y intercept and slope of the line are used to calculate the Km and Vmax for the experiment. The y intercept is equal to 1/Vmax , and the slope of the line is equal to Km/Vmax. In this case, a linear fit of the data points produced a line with the equation y = 2.61x + 6.48. Using the above equations, the Vmax and Km were calculated as 0.15 s᎑1 and 0.39% (or 110 mM) H2O2, respectively. The Km compares well with the published value of 25 mM for catalase (7), even though the demonstration only uses crude liver extract. Variations There are many possible variations of this demonstration. It can be expanded to include the kinetic analysis of a more dilute solution of the beef liver extract. Dilute the extract used in the demonstration 1:3 with ice-cold 0.1 M potassium phosphate buffer pH 7.0. Repeat the kinetic analysis by timing how long it takes for pieces of extract-soaked filter paper to rise to the top of the five different concentrations of H2O2. Plot the data on a Lineweaver–Burk plot and compare the Km and Vmax 1452
Table 1. Data and Calculated Values for Catalase Activity H2O2 concn (%)
Av Time/ s
V/s᎑1
1/H2O2 concn (%᎑1)
(1/V )/ s
2
7
0.14
0.5
7
1
9
0.11
1
9
0.083
2
12
0.5
12
0.3
15
0.065
3.33
15
0.2
19
0.052
5
19
Figure 1. Double reciprocal plot of catalase enzymatic activity.
of this reaction to those of the more concentrated enzyme solution. The addition of several higher and lower concentrations of H2O2 will allow the collection of a wider range of time points and allow the data to be plotted on a classic Michaelis– Menten velocity vs substrate concentration plot. In this case, however, a more advanced computer program such as Sigma Plot or Kalidagraph is needed to calculate the Km and Vmax. Another readily available source of catalase is potato. Although potato extract was a suitable substitute for liver extract, for unknown reasons, the calculated Km in that enzymatic reaction was consistently at least 25-fold higher than the published Km value of 25 mM for catalase (7). Disposal All solutions can be flushed down the drain with lots of water. Literature Cited 1. Hamilton, T. M.; Dobie-Galuska, A. A.; Wietstock, S. M. J. Chem. Educ. 1999, 76, 642. 2. Cornely, K.; Crespo, E.; Earley, M.; Kloter, R.; Levesque, A.; Pickering, M. J. Chem. Educ. 1999, 76, 644. 3. Helliwell, S.; Harden, T. J. J. Chem. Educ. 1996, 73, 368. 4. Bateman, R. C. Jr.; Evans, J. A. J. Chem. Educ. 1995, 72, A240. 5. Rowe, H. A.; Brown, M. J. Chem. Educ. 1988, 65, 548. 6. Choinski, J. S.; Patterson, J. W. J. Biol. Educ. 1993, 27, 7. 7. Mozaffar, S.; Ueda, M.; Kitatsuji, K.; Shimizu, S.; Osumi, M.; Tanaka, A. Eur. J. Biochem. 1986, 155, 527.
Journal of Chemical Education • Vol. 77 No. 11 November 2000 • JChemEd.chem.wisc.edu
