Enzymes are biological catalysts that speed up chemical reactions. Enzymes are specific and only speed up one type of reaction. The chemical reactants of an enzymatic reaction are called SUBSTRATES. Substrates bind to the active site of the enzyme. Once the reaction is complete, the PRODUCTS are released from the enzyme’s active site, and the enzyme can be reused to catalyze the next reaction.
Hydrogen peroxide is a metabolic waste product. High levels of hydrogen peroxide are toxic to cells and must be converted into less harmful substances. We will be working with the enzyme CATALASE. Catalase is found in a eukaryotic organelle, the peroxisome. Catalase accelerates the breakdown of hydrogen peroxide (H2O2) into water and oxygen. Catalase is an enzyme commonly found in ALL plant and animal cells as all cells can produce H2O2. The catalase used in this experiment was obtained from potatoes.
Using the reaction below, label the substrate, products, and enzyme.
Catalase
2H2O2 2H2O + O2
To determine the rate of activity of the enzyme, we will measure the bubbles that form during the reaction. The bubbles indicate that oxygen is being released. The more bubbles produced, the higher the activity rate of the enzyme.
Enzymes are proteins. Proteins are composed of amino acids and are folded into a unique shape. Extreme environmental changes may cause proteins to unfold (denature). Loss of protein shape causes a loss of function. Enzymes tend to function best under certain environmental conditions. These ideal environmental conditions vary among different enzymes and different species.
Lab safety and Supplies
- Goggles and gloves
- You will need a small ruler with a metric scale (cm and mm)
Practice: Measure the height of the bubble column (mm)
See PowerPoint file (Slide 6). Measure the height of each of the bubble columns (mm) in the three tubes.
| Tube number | Bubble Column Height (mm) |
| 1 | |
| 2 | |
| 3 |
Part One: Effect of Substrate on Catalase Activity
Procedure
- Three test tubes were labeled and marked at the 1 cm and 5 cm levels.
- Shake the bottle of catalase thoroughly to mix contents before dispensing into the test tubes.
- Tube 1
- Catalase was added to the 1 cm mark.
- Hydrogen peroxide was added to the 5 cm mark.
- Swirl to mix. Waited one minute for bubbles to develop.
- Tube 2
- Catalase was added to the 1 cm mark.
- Water was added to the 5 cm mark.
- Swirl to mix. Waited one minute for bubbles to develop.
- Tube 3
- Catalase was added to the 1 cm mark.
- Sucrose was added to the 5 cm mark.
- Swirl to mix. Waited one minute for bubbles to develop.
- PowerPoint file (Slide 7) shows the results of this experiment. Put the PowerPoint in Slide Show Mode (full screen) and use a ruler to measure the heights of the bubble columns of each tube. Record your data in Table 1.
Table 1: Effect of Substrate on Catalase Activity
| Tube | Contents | Bubble Column Height (mm) | Rate of Enzyme Activity: High, Medium, Low, or No activity |
| 1 | Catalase Hydrogen peroxide | ||
| 2 | Catalase Water | ||
| 3 | Catalase Sucrose |
- Which tube was the negative control? _____________
- The enzyme catalase works best with which substrate? (circle one)
Hydrogen peroxide Water Sucrose
Part Two: Effect of Temperature on Catalase Activity
Active sites of enzymes bring substrate molecules closer together and thus increase the chance that the molecules will interact. Generally, an increase in temperature tends to increase the movement of molecules in a system. A minimal increase in temperature causes an increase in the enzyme activity rate. This is due to more substrate collisions with the active site of the enzyme.
Read the experiment and formulate a hypothesis regarding the activity rate of the catalase enzyme under different temperatures. Formulate your hypothesis BEFORE looking at the results in the lab PowerPoint file. (Refer to specific temperatures rather than tube numbers in your hypothesis.)
Procedure
- Three test tubes were labeled and marked at the 1 cm and 5 cm levels.
- Shake the bottle of catalase thoroughly to mix contents before dispensing into the test tubes.
- Tube 1: Cold (4°C)
- Catalase was added to the 1 cm mark.
- BOTH tubes were placed in the ice bucket for 15 minutes.
- After 15 minutes the tubes were removed from the ice bucket and the cold hydrogen peroxide was added to the 5 cm mark to the tube with the enzyme.
- Swirl to mix. Waited one minute for bubbles to develop.
- Tube 2: Warm (37°C)
- Catalase was added to the 1 cm mark.
- Another test tube was obtained and marked at the 4 cm level. Hydrogen peroxide (H2O2) was added to the 4 cm mark
- BOTH tubes were placed in the 37°C waterbath for 15 minutes.
- After 15 minutes the tubes were removed from the waterbath and the warm hydrogen peroxide was added to the 5 cm mark to the tube with the enzyme.
- Swirl to mix. Waited one minute for bubbles to develop.
- Tube 3: Boiling (97°C)
- Catalase was added to the 1 cm mark.
- This tube was placed in the 97°C waterbath for 15 minutes.
- Using tongs, the tube was removed from the waterbath and the (room temperature) hydrogen peroxide (H2O2) was added to the 5 cm mark to the tube with the enzyme.
- Swirl to mix. Waited one minute for bubbles to develop.
- PowerPoint file (Slide 9) shows the results of this experiment. Put the PowerPoint in Slide Show Mode (full screen) and use a ruler to measure the heights of the bubble columns of each tube. Record your data in Table 2.
Table 2: Effect of Temperature on Catalase Activity
| Tube | Temperature | Bubble Column Height (mm) | Rate of Enzyme Activity: High, Medium, Low, or No activity |
| 1 | Cold (4°C) | ||
| 2 | Warm (37°C) | ||
| 3 | Very Hot (97°C) |
- The enzyme catalase had the highest rate of activity at which temperature? _________
- Do the results support the hypothesis? ___________
- The catalase enzyme solution used in this experiment was made from what source? ___________
- If warm temperatures tend to speed up enzymatic reactions, explain what happened in Tube 3. Speculate why Tube 1 yielded its rate of activity. (Hint: What is the source of the enzyme in this experiment?)
- Using the data in Table 2, create a bar graph. Plot temperature on the x-axis and height of the bubble column on the y-axis.
The Effect of Temperature on Enzyme Activity
Part Three: Effect of Enzyme Amount
Consider the check-out lanes at a grocery store. Assume that cashiers spend the same amount of time with each customer. The amount of time customers wait in line for a cashier is shorter if the store has several check-out lanes staffed with cashiers. If this example is related to an enzymatic reaction, then the customers can be considered the substrates and the cashiers are the enzymes.
What happens to the customer wait time for a cashier when there are several check – out lanes available compared to one check-out lane available?
Increase, Decrease, Stays the same (circle one)
Read the experiment and formulate a hypothesis regarding the rate of enzyme activity in samples that have different amounts of the enzyme. Formulate your hypothesis BEFORE looking at the results in the lab PowerPoint file. (Refer to specific amounts rather than tube numbers.)
Procedure
- Three clean test tubes were labeled.
- Shake the bottle of catalase thoroughly to mix contents before dispensing into the test tubes.
- Tube 1: No catalase enzyme
- Tube 2: 1 cm of catalase enzyme
- 1 cm of catalase and 4 cm of hydrogen peroxide were added to the tube.
- Swirl to mix. Waited one minute for bubbles to develop.
- Tube 3: 3 cm of catalase enzyme
- 3 cm of catalase and 4 cm of hydrogen peroxide were added to the tube.
- Swirl to mix. Waited one minute for bubbles to develop.
- PowerPoint file (Slide 11) shows the results of this experiment. Put the PowerPoint in Slide Show Mode (full screen) and use a ruler to measure the heights of the bubble columns of each tube. Record your data in Table 3.
Table 3: Effect of Enzyme Amount
| Tube | Concentration of Enzyme | Bubble Column Height (mm) | Rate of Enzyme Activity: High, Medium, Low, or No activity |
| 1 | 0 cm | ||
| 2 | 1 cm | ||
| 3 | 3 cm |
- Do the results support the hypothesis? _____________
- Do all the tubes contain the same amount of substrate? ___________
- If Tubes 2 and 3 are allowed to react for an unlimited amount of time, would there still be a difference in the final heights of the bubble columns? Explain your answer.
- If Tube 1 had significant bubbles, what would you conclude about this experiment and why?
Part Four: Effect of Substrate Amount
Consider the check-out lanes at a grocery store. Assume that cashiers spend the same amount of time with each customer. The amount of time customers wait in line for a cashier depends on how many check-out lanes are open and staffed. If this example is related to an enzymatic reaction, then the customers can be considered the substrates and the cashiers are the enzymes.
What happens to the customer wait time for a cashier when there are many customers and only one check- out lane available?
Increase, Decrease, Stays the same (circle one)
Read the experiment and formulate a hypothesis regarding the rate of enzyme activity in samples that have different amounts of the substrate. Formulate your hypothesis BEFORE looking at the results in the lab PowerPoint. (Refer to specific amounts rather than tube numbers.)
Procedure
- Three clean test tubes were labeled.
- Shake the bottle of catalase thoroughly to mix contents before dispensing into the test tubes.
- Tube 1: 1 cm of the H2O2 substrate
- Tube was marked at 1 cm and 2 cm levels.
- Catalase was added to the 1 cm level and hydrogen peroxide to the 2 cm level.
- Swirl to mix. Waited three minutes for bubbles to develop.
- Tube 2: 2 cm of the H2O2 substrate
- Tube was marked at 1 cm and 3 cm level.
- Catalase was added to the 1 cm level and hydrogen peroxide to the 3 cm level.
- Swirl to mix. Waited three minutes for bubbles to develop.
- PowerPoint file (Slide 13) shows the results of this experiment. Put the PowerPoint in Slide Show Mode (full screen) and use a ruler to measure the heights of the bubble columns of each tube. Record your data in Table 4.
Table 4: Effect of Substrate Concentration
| Tube | Concentration of Substrate (H2O2) | Bubble Column Height (mm) After 3 minutes | Rate of Enzyme Activity: High, Medium, Low, or No activity |
| 1 | 1 cm | ||
| 2 | 2 cm |
- Do the results support the hypothesis? _____________
- Do the tubes contain the same amount of substrate? ___________________
- Do the tubes contain the same amount of enzyme? _______________
- Does increasing the amount of substrate produce more enzyme activity? _______ Explain why or why not.
Part Five: Effect of pH on Catalase Activity
Read the experiment and formulate a hypothesis regarding the activity rate of the potato catalase enzyme under different pH conditions. Formulate your hypothesis BEFORE looking at the results in the lab PowerPoint file. (Refer to specific pH conditions rather than tube numbers in your hypothesis.)
Procedure
- Six clean test tubes were labeled.
- Shake the bottle of catalase thoroughly to mix contents before dispensing into the test tubes.
- Tube 1: Water
- 1cm of catalase was added to the tube.
- 2 cm of water was added to the tube. Waited one minute to allow the enzyme to adjust to the solution.
- 4 cm of hydrogen peroxide was added to the tube.
- Swirl to mix. Waited one minute for bubbles to develop.
- Tube 2: pH 3
- 1cm of catalase was added to the tube.
- 2 cm of pH 3 solution was added to the tube. Waited one minute to allow the enzyme to adjust to the pH solution.
- 4 cm of hydrogen peroxide was added to the tube.
- Swirl to mix. Waited one minute for bubbles to develop.
- Tube 3: pH 5
- 1cm of catalase was added to the tube.
- 2 cm of pH 5 solution was added to the tube. Waited one minute to allow the enzyme to adjust to the pH solution.
- 4 cm of hydrogen peroxide was added to the tube.
- Swirl to mix. Waited one minute for bubbles to develop.
- Tube 4: pH 7
- 1cm of catalase was added to the tube.
- 2 cm of pH 7 solution was added to the tube. Waited one minute to allow the enzyme to adjust to the pH solution.
- 4 cm of hydrogen peroxide was added to the tube.
- Swirl to mix. Waited one minute for bubbles to develop.
- Tube 5: pH 9
- 1cm of catalase was added to the tube.
- 2 cm of pH 9 solution was added to the tube. Waited one minute to allow the enzyme to adjust to the pH solution.
- 4 cm of hydrogen peroxide was added to the tube.
- Swirl to mix. Waited one minute for bubbles to develop.
- Tube 6: pH 12
- 1cm of catalase was added to the tube.
- 2 cm of pH 12 solution was added to the tube. Waited one minute to allow the enzyme to adjust to the pH solution.
- 4 cm of hydrogen peroxide was added to the tube.
- Swirl to mix. Waited one minute for bubbles to develop.
- PowerPoint file (Slide 15) shows the results of this experiment. Put the PowerPoint in Slide Show Mode (full screen) and use a ruler to measure the heights of the bubble columns of each tube. Record your data in Table 5.
Table 5: Effect of pH on Catalase Activity
| Tube | pH | Bubble Column Height (mm) | Rate of Enzyme Activity: High, Medium, Low, or No activity |
| 1 | Water | ||
| 2 | 3 | ||
| 3 | 5 | ||
| 4 | 7 | ||
| 5 | 9 | ||
| 6 | 12 |
- Do the results support the hypothesis? _____________
- Explain the purpose of Tube 1.
- The enzyme catalase had the highest rate of activity at which pH? _________
- Speculate on why this pH (see question 1) had the highest rate of activity.
Clean up
- All labels and marks were removed from the test tubes by cleaning them with alcohol wipes.
- All test tubes were cleaned at a lab sink with warm water, soap and test tube brushes.
- Clean tubes were placed upside down in the test tube racks to drip dry.
- Test tube racks were placed on the absorbent pad in the back of the lab.
- Stock solutions were returned to their appropriate storage containers.
Review Questions
- If another substance competed with hydrogen peroxide for the active site on catalase, how would the rate of enzyme activity be affected? (increase, decrease, or stay the same) (circle one)
- Lipase is an enzyme that helps digest fat droplets in the small intestine. This enzyme requires a slightly basic pH in order to work properly. Do you think this enzyme would work properly in your stomach? Why or why not?
- Lactose is a disaccharide found in milk products. In our digestive system, lactose will break down into glucose and galactose (both monosaccharides) in the presence of water and lactase.
Using the underlined components, write the chemical reaction (in words) for lactose digestion below.
- Label the substrate(s), product(s), and enzyme(s).
- Once the lactose breaks apart, what will happen to the lactase molecule?
- Note that water is involved in breaking down lactose. Therefore, this is an example of what type of reaction?
Do you have a similar assignment and would want someone to complete it for you? Click on the ORDER NOW option to get instant services. We assure you of a well written and plagiarism free papers delivered within your specified deadline.