Enzyme Activity
OVERVIEW: In this laboratory you will use the enzyme, peroxidase, from turnips to study enzyme activity and factors that affect it. Peroxidase will catalyze a reaction in which a reducing agent, guaiacol, changes color when it is oxidized by H2O2. This color change will be measured by using a spectrophotometer or colorimeter connected to a CBL data recorder to determine enzyme activity.
peroxidase
Guaiacol + 2 H2O2 ------à tetraguaiacol + 8 H2O
(colorless) (brown)
OBJECTIVES: At the completion of this laboratory you should be able to:
- graph data from an enzyme experiment
- determine the rates for enzymatically catalyzed reactions
- discuss a method for determining enzyme activity
- discuss the relationship between dependent and independent variables
- discuss the effect on initial reaction rates produced by changes in temperature, pH, enzyme concentration, and substrate concentration
- design an experiment to measure the effect on enzyme activity produced by changes in temperature, pH, enzyme concentration, and substrate concentration
INTRODUCTION: Much can learned about enzymes by studying the kinetics (changes in rate) of enzyme-catalyzed reactions. For example, it is possible to measure the amount of product formed, or the amount of substrate used, from the moment the reactants are brought together until the reaction has stopped. If the amount of product formed is measured at 30-second intervals and this quantity is plotted on a graph, the curve shown below is obtained.
Observe the solid line for this reaction. At time 0 there is no product. After 30 seconds 5 micromoles have been formed; after 1 minute, 10; after 2 minutes, 20. The rate of this reaction could be given as 10 micromoles of product formed per minute for this initial time period. Note however, that by the third and fourth minutes, only about 5 additional micromoles of product have been formed. During the first 3 minutes the rate is constant. From the third minute through the eighth minute, the rate is changing---it is slowing down. For each successive minute after the first 3 minutes, the amount of product formed in that interval is less than in the preceding minute. From the seventh minute onward, the reaction rate is very slow.
For comparison of kinetics of one reaction with another, a common reference point is needed. For example, suppose you wanted to compare the effectiveness of peroxidase obtained from a turnip with that of peroxidase obtained from liver. Would you want to compare the two reactions during the first few minutes when the rate is constant or later when the rates are changing? It is best to compare the reactions when the rates are constant. In the first few minutes of an enzymatic reaction such as this, the number of substrate molecules is usually so large compared to the number of enzyme molecules that changing the substrate concentration does not (for a short period at least) affect the number of successful collisions between the substrate and enzyme. During this early period, the enzyme is acting on substrate molecules at a constant rate. The slope of the graph line during this early period is called the “initial velocity of the reaction.” The initial velocity (or rate) of an y enzyme-catalyzed reaction is determined by the characteristics of the enzyme molecule. It is always the same for an enzyme and its substrate as long as temperature and pH are held constant and the substrate is present in excess.
The rate of the reaction is therefore the slope of the linear portion of the curve. To determine a rate, pick any two points on the straight-line portion. However, do not use actual data points…use points that fall on the line. The slope of the line is expressed as:
ΔY product2 – product1
ΔX t2 – t1
PROCEDURE: The turnip extract containing the peroxidase enzyme has been prepared for you. One gram of turnip was placed in a blender with 200 mL of distilled water and blended thoroughly.
1. Prepare 3 test tubes as follows:
Tube Tube Tube
#1 #2 #3
guaiacol 0.1 mL 0.1 mL ---
H2O2 --- 0.2 mL ---
turnip extract 1.0 mL --- 1.0 mL
(peroxidase)
distilled water 5.2 mL 5.0 mL ---
NOTES: 2 drops = 0.1 mL
Tube #1 is the BLANK & is only used for calibrating the spec. – step 2 below.
2. Set the wavelength on the Spectrophotometer to 500nm. Use the knob on the left (that turned the spec “on”) to adjust the meter to 0% T without a test tube in the chamber. Then place test tube #1 in the chamber and use the right hand knob to adjust the meter to 100% T.
3. Pour the contents of tube #2 into tube #3, then quickly cover with parafilm & invert 2X to mix the reagents. Start timing as soon as the tubes are mixed because the reaction has begun.
4. Wipe the outside of the tube & place in the spec chamber to begin recording.
5. Take the first measurement 10 seconds after the tubes were mixed & record at 10 second intervals for 2 minutes
Tomorrow you will design your own experiment testing one of the following conditions. You will need to run a control/baseline (what you did today) and then modify the conditions accordingly to determine the effects on enzyme activity. Please select from the following.
1. Effect of varying the amount of enzyme: Run a standard and then vary the concentration of enzyme to see how the reaction rate is affected. Suggested concentrations are 0.5X, 2X, & 3X the amount of enzyme. Each time you change the amount enzyme you must offset the difference by changing the amount of distilled water. Repeat each concentration for a better experimental design.
2. Effect of varying substrate concentration (H2O2): Run a standard and then vary the amount of H2O2. Suggested concentrations are 0/5X, 2X & 3X the amount of substrate. Each time you change the amount substrate you must offset the difference by changing the amount of distilled water. Repeat each concentration for a better experimental design.
3. Effect of temperature: Run a standard at room temperature set up an experiment to test at least 3 temperatures between 0˚C and 40˚C. Make sure you place cuvettes #2 & #3 in the water bath for at least 5 minutes before you mix them.
4. Effect of pH: Measure the effect of pH by using buffers instead of water. Be sure to use a range of pH buffers that spans acidic and basic conditions. See your instructor to see what is available.
5. Effect of heat: Heat a sample of turnip extract in a boiling water for varying amounts of time. Suggested times include 1 minute, 3 minutes, 5 minutes and 10 minutes. Cool the extract to room temperature before mixing with the other ingredients
Lab Write-up info:
Hypothesize what will happen in your experiment
Data Analysis: When you have completed you experiment, prepare a graph on which you plot absorbance on the y-axis and time on the x-axis. Invert the y-axis so that 100% T is at the X-Y vertex. Plot each set of data on 1 graph and draw a “best fit” smooth, continuous line. Use the initial slope of your line (not the actual data points) to calculate the rate under each of the different conditions.
Discussion questions
Did the data support or refute your hypothesis? Why or why not?
ENZYME ACTIVITY
Preparing the CBL for using the COLORIMETER probe in CHANNEL 1
1. Turn CBL ON.
2. Press PRGM
3. Select 1:DataMate by pressing ENTER – 2X (There may be a message stating “Continue with interface.” Select 1:Yes
4. Press 1:Setup
5. Press CLEAR (The CBL will begin “Checking sensors” & then show CH1 as empty.)
6. Press ENTER (This will show all sensors.)
7. Press 7:more
8. Press 2:colorimeter
9. Move cursor to MODE
10. Press 2:Time graph
11. Press 2:Change time settings
12. Set “Time between samples” as 10
13. Set “Number of samples” as 9 (This will take 9 readings at 10 second intervals.)
14. Press 1:OK
15. Press 1:OK