Enzyme activity lab
Purpose: How do environmental factors like temperature and pH affect the speed of enzyme
interaction with substrate chemicals?
Background:
Enzymes are biological catalysts. They speed up chemical reactions in biological systems by
lowering the activation energy, or the energy needed for molecules to begin reacting with each other.
Enzymes do this by colliding with a substrate molecule (forming an enzyme-substrate complex).
Enzymes have specific 3-D shapes that determine their functions. The enzyme’s active site is very
specific, allowing only a particular chemical substrate to bind there.
Some environmental factors can break chemical bonds that hold the enzyme in its particular 3-D
shape. This is called denaturation, and when it occurs the enzyme is no longer able to speed up the
reaction of its chemical substrate.
To begin this investigation, you will focus on the enzyme peroxidase obtained from a turnip, one of
numerous sources of this enzyme. Peroxidase is one of several enzymes that break down peroxide
(a potentially dangerous chemical produced during normal cell activity). The break down chemical
reaction is:
peroxidase
hydrogen peroxide
2H2O2
water and oxygen
2H2O +
O2
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 this experiment, we will use an indicator chemical that
detects the presence of the oxygen product. The chemical guaiacol binds instantly with oxygen to
form tetraguaiacol, which is brownish in color.
guaiacol + O2
(clear)
tetraguaiacol
(brown)
So we now have a basic way to measure peroxidase enzyme speed of breaking down its hydrogen
peroxide substrate – the faster the solution turns brown, the faster the enzyme must be breaking
down substrate to form oxygen product.
Learning Objectives:

To understand the relationship between enzyme structure and function.

To make some generalizations about enzymes by studying just one enzyme in particular.

To determine how temperature and pH change the rate of chemical reactions.
Procedure 1: (enzyme speed with no modified environmental factors)
Materials:
• Turnip peroxidase (~ 4% suspension of
turnip cells blended with distilled water)
• Timer
• 0.1 % hydrogen peroxide
• Funnel
• 0.3 % guaiacol
• Safety goggles
• Syringes
• Distilled water
• Test tubes and test tube rack
Step 1: Using two test tubes, mark one “substrate” and the other tube “enzyme.” To the substrate
tube, add 7 mL of distilled water, 0.3 mL of 0.1 percent hydrogen peroxide, and 0.2 mL guaiacol for a
total volume of 7.5 mL. Shake the test tube a bit to gently mix.
Step 2: To the enzyme tube, add 6.0 mL of distilled water and 1.5 mL of peroxidase for a total
volume of 7.5 mL. Shake the test tube to gently mix.
Step 3: Combine the contents of the two tubes (substrate and enzyme) by pouring one tube into
another. Shake the test tube to gently mix. Immediately begin timing the reaction.
Step 4: Observe the color change for the next 5 minutes. Record the observed color at 0, 1, 2, 3, 4,
and 5 minutes. You could potentially take a picture of the test tube at each minute with a cell phone
camera, or just use the color palette / chart to estimate the number color at each time period.
Review your group and class results – do we need to take measurements every minute for 5 minutes,
or could we use less time to estimate enzyme speed? We can decide as a class.
Procedure 2: (enzyme speed with modified environmental factors)
Materials:
• Turnip peroxidase (~ 4% suspension of
turnip cells blended with distilled water)
• Syringes
• 0.1 % hydrogen peroxide
• Buffer solutions of pH 3, 5, 6, 7, 8, 10
• 0.3 % guaiacol
• Hot plates / ice
• Distilled water
• Beakers with water
• Test tubes and test tube rack
• Thermometers on ring stands
• Timer
• Safety goggles
• Funnel
Some classes – pH modification
Step 1: Label pairs of test tubes as a substrate or enzyme test tube. This time you will substitute a
different pH buffer for the distilled water used in the original enzyme tubes. Prepare the tubes as
follows and be sure to label them.
•
For each substrate tube in a pair, add 7 mL of distilled water (pH solution only goes in enzyme
tube), 0.3 mL of hydrogen peroxide, and 0.2 mL of guaiacol for a total volume of 7.5 mL.
•
For each enzyme tube in the pair, add 6.0 mL of a specific pH solution and 1.5 mL of
peroxidase for a total volume of 7.5 mL. Use the pH solution available at your group’s table.
Use the dH2O syringe to load your pH solution. In between each use, draw up 6 mL of dH 2O
and flush into the waste cup to clean the syringe.
•
Shake each to gently mix.
Step 2: Combine the substrate and enzyme tube pairs when ready to test. Shake to gently mix and
begin timing the reactions.
Step 3: Record the observed color for each tube at 0 minutes each minute for ___ minutes (however
long we decided from procedure 1). Again, a cell phone and/or camera are excellent ways to record
color change.
Step 4: Use the palette/color chart to help you quantify the changes you observe.
Some classes – temperature modification
Step 1: Label pairs of tubes substrate and enzyme. Fill tubes as in procedure 1.
Substrate tube: add 7 mL of distilled water, 0.3 mL of 0.1 percent hydrogen peroxide, and 0.2
mL guaiacol for a total volume of 7.5 mL. Shake the test tube a bit to gently mix.
Enzyme tube: add 6.0 mL of distilled water and 1.5 mL of peroxidase for a total volume of 7.5
mL. Shake the test tube to gently mix.
Step 2: Place the enzyme and substrate tubes into a water bath of a specific temperature (use the
water bath at your group’s table). Leave them in there for 4 – 5 minutes.
Step 3: Be prepared to mix the tubes and start timing for 5 minutes as soon as you pull the test
tubes out of the water bath. You could also put the mixed reacting tube back in the water bath to
keep the solution at that temperature as well (do NOT do this with very high temperature water baths
– we don’t want you to burn yourself).
Step 4: Record the observed color for each tube at 0 minutes each minute for ___ minutes (however
long we decided from procedure 1). Again, a cell phone and/or camera are excellent ways to record
color change.
Step 5: Use the palette/color chart to help you quantify the changes you observe.