AP Biology Investigation #13: Enzyme Activity

AP Biology | Mr. Austin

Background:
Peroxide (such as hydrogen peroxide) is a toxic byproduct of aerobic (oxygen-using) metabolism. Peroxidase is
an enzyme that breaks down these peroxides. It is produced by most cells in their peroxisomes.

• The general reaction of an enzyme can be depicted as follows:

• For this investigation the specific reaction is as follows:

• Overall chemical equation:

Notice that the peroxidase is present at the start and end of the reaction. Like all catalysts, enzymes are not
consumed by the reactions. To determine the rate of an enzymatic reaction, you must measure a change in
the amount of at least one specific substrate or product over time. In a decomposition reaction of peroxide by
peroxidase (as noted in the above formula), the easiest molecule to measure would probably be oxygen, a
final product. This could be done by measuring the actual volume of oxygen gas released or by using an
indicator. In this experiment, an indicator for oxygen will be used. The compound guaiacol has a high affinity
for oxygen, and in solution, it binds instantly with oxygen to form tetraguaiacol, which is brownish in color.
The greater the amount of oxygen gas produced, the darker brown the solution will become.

Questions:
1. How fast does peroxidase break down peroxides under neutral conditions?
2. At what pH does peroxidase function the fastest?
AP Biology Investigation #13: Enzyme Activity
AP Biology | Mr. Austin

Part 1: Measuring peroxidase activity in plant material and determining a baseline

Materials
• Spectrophotometer • Guaiacol • pH 7 buffer solution
• 2 cuvettes • Turnip peroxidase • Parafilm
• 3 syringes (2.5 mL) solution • Disposable pipet
• 1 syringe (10 mL) • Dilute hydrogen
• 2 test tubes peroxide

Procedures
1. Turn on your spectrophotometer so that it will warm up appropriately. Set the wavelength to 436 nm.
2. Using tape, mark one test tube “s” for substrate and the other test tube “e” for enzyme.
3. To the substrate tube, add 2 mL of dilute hydrogen peroxide, 1 mL of guaiacol, and 1 mL of neutral
buffer (pH 7) for a total volume of 4 mL. Mix the test tube, being careful not to spill its contents.
4. To the enzyme tube, add 1 mL of turnip peroxidase solution and 3 mL of neutral buffer for a total
volume of 4 mL. Mix the test tube, being careful not to spill its contents.
5. Set the two test tubes aside.
6. Set the spectrophotometer to zero absorbance (calibrate) by using a blank cuvette containing all the
appropriate materials except the substrate (i.e. 1 mL of guaiacol, 1 mL of turnip peroxidase, and 4 mL
of neutral buffer)
7. Transfer the mixture from the substrate test tube to the enzyme test tube. Cover with parafilm and
invert twice to mix.
8. Using a disposable pipet, quickly add contents to a new clean cuvette after mixing. Place the cuvette
into the spectrophotometer and record the absorbance. This is your initial time reading (0 minutes).
9. Remove the cuvette and rerecord the absorbance every minute for five minutes. Be sure to gently mix
the cuvette and wipe its surface with a cleaning wipe before each reading. Cover cuvette with lid to
avoid spilling.
10. Graph the absorbance over the 5 minute interval and calculate the rate of the enzymatic reaction
(absorbance/minute) for the baseline conditions of this experiment.

Data
Time 0 min 1 min 2 min 3 min 4 min 5 min
Absorbance
Part 2: Investigating variables that affect the rate of enzyme reaction

Materials
• Spectrophotometer • 3 syringes (2.5 mL) • Turnip peroxidase
• 6 cuvettes • 1 syringe (10 mL) solution
• 12 test tubes • Guaiacol • Dilute hydrogen
peroxide

Procedures
1. Turn on your spectrophotometer so that it will warm up appropriately. Set the wavelength to 436 nm.
2. Label 6 clean test tubes “s” for substrate and label another 6 clean test tubes “pH 3”, “pH 5”, “pH 6”,
“pH 7”, “pH 8”, and “pH 10”.
3. To the six substrate tubes, add 2 mL dilute hydrogen peroxide, 1 mL guaiacol, and 1 mL neutral buffer
(pH 7) for a total volume of 4 mL in each tube. Mix the test tube, being careful not to spill its contents.
4. Into tube labeled “pH 3,” add 1 mL of turnip peroxidase solution and 3 mL of pH 3 solution for a total
volume of 4 mL. Mix the test tube, being careful not to spill its contents.
5. Repeat step 4 for the tubes labelled pH 5, 6, 7, 8, and 10 with their respective pH solutions.
6. Set the spectrophotometer to zero absorbance (calibrate) by using a blank cuvette containing all the
appropriate materials except the substrate (i.e. 1 mL of guaiacol, 1 mL of turnip peroxidase, and 4 mL
of neutral buffer)
7. Transfer the mixture from a substrate test tube to the test tube labelled pH 3. Cover with parafilm and
invert twice to mix.
8. Using a disposable pipet, quickly add contents to a new clean cuvette after mixing. Place the cuvette
into the spectrophotometer and record the absorbance. This is your initial time reading (0 minutes).
9. Remove the cuvette and rerecord the absorbance again at a time you choose as optimal based on the
results obtained in Part 1. Be sure to gently mix the cuvette and wipe its surface with a cleaning wipe
before the next reading. Cover cuvette with lid to avoid spilling.
10. Repeat steps 7-9 for the remaining pH buffer solutions.
11. Calculate the rate of reaction for each pH solution (absorbance/min). Graph your rate results relative
to pH.
Data
pH Initial Absorbance Final Absorbance Reaction Rate
(0 min) ( ___ min) (absorbance/min)
3

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