Lab AP2 Enzyme Catalysis Date
Student name Lab Partner: name AP Biology, Period X
Background:
Many chemical reactions occur spontaneously but they may occur too slowly to detect. The reason for this is that there is a certain amount of energy that is required for starting the reaction, the activation energy. Addition of a catalyst, a chemical that is not consumed in the reaction, speeds up the reaction. An enzyme is a protein that functions as a catalyst by lowering the activation energy.
Enzymes are important in metabolism because without them, the many chemical reactions taking place would be slowed and metabolic pathways would be congested. Enzymes do not change the free-energy of the reaction and can not change the type of reaction from one that absorbs energy (endergonic) to one that releases energy (exergonic). Enzymes speed up a reaction allowing the metabolic reactions to occur in a more regulated and orderly manner.
The chemical that an enzyme acts on is the substrate which is very specific to the enzyme. The enzyme and substrate join to form a complex during which time, the catalytic action of the enzyme converts the substrate to the product. The specificity of the enzyme is due to the unique shape of the protein. There is a region of the enzyme, called the active site, which binds to the substrate. Upon completion of the reaction, the enzyme is released virtually unchanged to then be able to take part in another reaction with a substrate molecule.
The rate at which an enzyme converts a substrate is a function of the concentration of the substrate and the speed at which the active site can convert substrate to product. Increasing the amount of substrate will increase the rate up to the point where all of the active sites of the enzyme are occupied at which point the enzyme is saturated. The only way to increase the rate of the reaction now is to add additional enzyme.
An enzyme can be affected by temperature and pH as well as by other compounds that specifically affect the enzyme. In general, increasing the temperature will increase the rate of the reaction catalyzed by the enzyme. Above a certain temperature, however, the speed of the reaction drops because the increasing temperature disrupts the molecular configuration of the enzyme. Thus each enzyme has an optimal temperature at which its reaction rate is greatest. Ultimately, the temperature may be so great that the enzyme, which is a protein, becomes denatured. Similar to temperature, enzymes have an optimal pH at which they function at the greatest reaction rate.
Purpose:
The purpose of the Enzyme Catalysis lab is to observe the reaction between hydrogen peroxide (H2O2) and the enzyme catalase producing water and oxygen and to measure the amount of oxygen that is created and to calculate the rate of the enzyme-catalyzed reaction. After performing this lab, we will be able to measure how changes in temperature, pH, enzyme concentration, and substrate concentration affect the reaction rates of an enzyme-catalyzed reaction in a controlled experiment. We’ll also be able to explain the effect of environmental factors on enzyme-catalyzed reactions. In part 2B, we will determine the initial amount of H2O2 in a 1.5% solution. In part 2D, we will determine the decomposition rate of 1.5% H2O2 when catalyzed by the catalase extract.
Procedure: For diagram of procedure, see Scientific Lab Notebook
Part 2A: Catalase Activity Demonstration
Observe the teacher demonstration and record observations and reactions.
Part 2B: The Base Line Assay
1) Measure 10 mL of 1.5% H2O2 with a 10 mL plastic graduated cylinder and pour H2O2 into a clean glass beaker.
2) Using a plastic 1 mL pipette, add 1 mL of distilled H2O to the H2O2. The H2O is used here instead of the enzyme solution.
3) Using a glass 10 mL graduated cylinder, measure 10 mL of 1.0 M H2SO4 and add to the beaker with the H2O and H2O2.
4) Mix this solution well. This is the Stock Base Line Solution.
5) To begin the assay portion, use a plastic pipette to add 5 mL of your Stock Base Line Solution to an empty 125 mL Erlenmeyer flask.
6) Record Initial burette reading.
7) Place a white square of paper on the burette stand’s base. Place the Erlenmeyer flask under the burette and titrate with KMnO4 until a persistent pink color is obtained. The solution will pass through yellow-brown color stage immediately before the pink. Once this yellow-brown stage is reached, use half-drops to obtain the pink color. While titrating, gently swirl the flask, not too much so that the solution becomes aerated.
8) Record Final burette reading.
9) Compare your final Base Line value with at least 2 other groups.
Part 2D: An Enzyme-Catalyzed Rate of H2O2 Decomposition
a) The Reaction
1) Measure 10 mL of 1.5% H2O2 with a plastic graduated cylinder and pour into seven clean 50 mL beakers (10 mL in each). For the interest of time, however, only use three beakers.
2) Label each beaker with the different amounts of time (10 seconds, 30 seconds, and 60 seconds).
3) Measure 10 mL of H2SO4 with a 10 mL glass graduated cylinder. Don’t add it yet, but keep it near to stop the reaction.
4) Using a 1 mL plastic pipette, add 1 mL catalase extracted from liver to each beaker. Swirl. The catalase extract must be kept on ice when not in use.
5) After the reaction time designated on the beaker (10 seconds, 30 seconds, and 60 seconds) is up, add the 10 mL of H2SO4. Swirl.
Note: We found it easiest to just do one reaction at a time. Add the catalase extract, allow to react, and then add H2SO4. Then re-measure the H2SO4 in the graduated cylinder and repeat for the 30 and 60 second reaction times.
b) The Assay
1) Using a new 1 mL plastic pipette, measure 5 mL of the solution into a 125 mL Erlenmeyer flask
2) Record Initial measurement on the titration burette.
3) Like in Part 2B, place the flask on a white piece of paper and titrate the solution with KMnO4 until the persistent pink color appears. Gently swirl the solution while titrating.
4) Record the Final measurement on the titration burette.
5) Repeat steps 1-4 of the Assay for each sample solution. Since there are so many people in the class and not enough flasks, after each titration, rinse the flask with tap water a few times then at least once with distilled water. If there is a precipitate on the bottom of the flask, pour some H2O2 in (just enough to cover the bottom of the flask) and swirl until dissolved. Then rinse 10 times with tap water and once with distilled water.
Data Tables and Graphs:
Part B:
Base Line CalculationsTrial 1 / Trial 2
Initial reading (mL) / 0.00 / 4.80
Final reading (mL) / 4.70 / 9.70
Base Line (mL) / 4.70 / 4.90
Part D:
Room Temperature Enzyme-Catalyzed ReactionKMnO4 (mL) / Time (seconds)
10 / 30 / 60 / 90 / 120 / 180 / 360
Base Line / 4.70 / 4.70 / 4.70 / 4.70 / 4.70 / 4.70 / 4.70
Initial Reading / 9.75 / 13.75 / -- / -- / -- / -- / --
Final Reading / 13.75 / 17.4 / -- / -- / -- / -- / --
│KMnO4 Consumed│ / 4.00 / 3.65 / 1.60* / 1.20* / .900* / .600* / .100*
│H2O2 Used│ / .700 / 1.05 / 3.10 / 3.50 / 3.80 / 4.10 / 4.60
* = Dry data, not actually obtained in our experiment.
Conclusion Question #3:
Rates of Reaction of H2O2Time (Seconds)
0 to 10 / 10 to 30 / 30 to 60 / 60 to 90 / 90 to 120 / 120 to 180 / 180 to 360
Rates Part B (mL/sec) / 7.00E-02 / 1.75E-02 / 6.83E-02 / 1.33E-02 / 1.00E-02 / 5.00E-03 / 2.80E-03
Calculations:
Base Line Calculations (Part 2B):
Base Line = Final reading – Initial reading
Base Line = 4.70E+00mL – 0.00E+00mL
Base Line = 4.70E+00mL
Part 2D: (Calculations Using values from the 10 second reaction)
KMnO4 Consumed calculations:
KMnO4 Consumed = Final Reading – Initial Reading
KMnO4 Consumed = 1.375E+01mL - 9.75E+00mL
KMnO4 Consumed = 4.00E+00mL
H2O2 Used Calculations:
H2O2 Used = │Baseline - KMnO4 Consumed │
H2O2 Used = │4.70E+00mL - 4.00E+00mL │
H2O2 Used = │7.00E-01mL │
H2O2 Used = 7.00E-01mL
Conclusion Question #2: Rates of Reaction of H2O2 (This example uses data from the 10-30 second times
Rate = ∆y / ∆x
Rate = (y-final – y-initial) / (x-final – x-initial)
Rate = (1.05E+00mL - 7.00E-01mL) / (30 sec – 10 sec)
Rate = 3.5E-01mL / 20 sec
Rate = 3.5E-01/20 mL/sec
Rate = 1.75E-02 mL/sec
Analysis:
In the Enzyme catalysis lab, I found that the amount of H2O2 initially present in a 1.5% solution is about 4.70E+00 mL. In Part D of this lab, I found that the rate at which H2O2 was consumed in an enzyme-catalyzed reaction was much faster in the beginning than towards the end. In the 10 second long reaction solution, the amount of H2O2 that was used was 7.00E-01 mL, giving it a rate in the first 10 seconds of about 7.00E-02 mL/sec. This rate at the beginning was higher than the rate at the end, which was 2.78E-03 mL/sec. This difference in rate shows how the reaction, if we had been measuring it continuously, was much faster in the beginning because there was more H2O2 available for the enzyme to react with. As we observed in the toothpick exercise, it was easy to pick up a toothpick out of a large pile and so we, the enzymes, broke them at a higher rate. Once the pile of toothpicks began to get smaller, it became more difficult to pick out one single toothpick and the rate at which we broke them was lower. Part A of the lab demonstrated how temperature had an effect on the reaction rate. The catalase extract that had been micro waved for 1 minute did not react at all with the H2O2, while the catalase that was at room temperature caused a large amount of bubbles to fizz out of the test tube instantaneously.
My experiment had errors. While I was titrating the solution, a drop or two splashed onto the inside wall of the Erlenmeyer flask. This meant that the amount of KMnO4 that the burette measured was greater than the amount that actually went into the solution. This error probably made the amount of H2O2 that was consumed seem greater than it actually was. Another error was that I wasn’t able to get the solution to stop exactly on the pink stage of the titration. What I measured was the milliliter reading on the burette once the solution showed a lingering yellow-brown color. Then, I added between ½ a drop and 2 drops and the solution turned into the murky brown stage with a heavy precipitate, indicating that I had titrated too far. So, while the measurements were close, they are a little bit off. Finally, one last error was that I wasn’t able to finish titrating all of the solutions, I only got through the 10 second and 30 second reactions. This meant that I had to use the dry data which was gathered under possibly different circumstances (i.e. Different temperature) or it was mixed differently. I used the dry set of data combined with my personal set of data to come up with one new set of data. The ratios could be off, especially where the personal data ends and the dry data begins. In the Amount of H2O2 Used in Different Timed Reactions graph, you can see a large difference between the 30 and 60 second values. This is where the data sets switch. If the 60 second value were just a tiny bit larger, or if the 30 second value were just a tiny bit smaller, then the ratio would be pushed greater than that of the 0 to 10 second one. This would be wrong because the enzyme reaction should have the highest rate when it’s just starting to react, not somewhere in the middle.
Conclusion:
The Enzyme Catalysis lab experiment was successful because it proved how the reaction rate at the beginning of the reaction was greater than at any other time during the reaction. The reaction rate of 7.00E-02 mL/sec was the largest rate of reaction, telling us how the enzyme was able to decompose the H2O2 much more efficiently when there was a larger supply of H2O2 molecules to decompose. Once the H2O2 molecules became scarcer, the enzyme reaction slowed down slightly because the active sites weren’t always being replenished right away. Part A demonstration was very successful because it showed how an enzyme that had been heated up too much had become denatured and no longer reacted with the H2O2.
During this lab, I became more skilled at titrating. I practiced trying to get half-drops to fall off of the end of the burette before the drop got too big. I also learned a lot about how enzymes work and function. Yes, I had been exposed to them in notes, but performing this lab experiment helped me a lot in understanding how they speed up a reaction.
In the future, this lab would be much more effective if there was more time allotted to perform more titrations. I understand that the quarter system of school and the limited amount of time in each class makes it more difficult to spend large amounts of time on single labs, but it might be a little less frustrating if there’s not so much of a rush to try and get things done.