Catalase Lab

Effects of Enzyme Catalysis of H2O2 by Catalase

Report by: Timmy Lin (#269164729)
October 17, 2011
Mr. Rienzi AP Biology

Problem: Measuring the effects of Catalase enzymes on hydrogen peroxide decomposition. Measuring the rate of the reaction when hydrogen peroxide and Catalase are mixed at the same ratio for different time (10, 20 30 60 120 180 360 seconds).

Background: Enzymes are biological catalysts that carry out cellular metabolic processes with the ability to enhance the rate of reaction between. They are large proteins made up of several hundred chains of amino acid. In an enzyme-catalyzed reaction, the substance to be acted upon, or substrate, binds to the active site of the enzyme. The enzyme and substrate are held together
in an enzyme-substrate complex by hydrophobic bonds, hydrogen bonds, and ionic bonds. Often, enzymes are multi-protein complexes made up of a number of individual protein subunits.
Hydrogen peroxide in lower concentrations, such as the types found in many homes, it works well as a disinfectant and antiseptic. Another use for hydrogen peroxide is to purify water. It is highly soluble in water, and adding it to water can increase oxygen and help eliminate dangerous contaminants. It is also used in some swimming pools in place of other water purifiers. Hydrogen peroxide is a colorless liquid that goes through the following spontaneous decomposition reaction.
2H2O2 2H2O + O2
This is a spontaneous reaction meaning it requires no energy to begin down but the reaction itself takes a long time. The enzymes lower the activation energy of the reaction allowing the reaction is occur at much faster rate allowing us to see the oxygen bubbles that are formed as a product of the reaction. The enzyme does this with the use of substrate conversion within the active site. The solution is then left there for a certain amount of time before it is denaturized. The denaturalization of enzyme can be done in many different ways; we stopped the reaction with the addition of Sulfuric Acid (H2SO4). The change in pH alters the enzymes quaternary structure and alters the active site so that it cannot accept H2O2 a substrate and ends catalysis. Other methods in the denaturalization of enzymes include altering salt concentration and temperature.
Hypothesis: If I leave the hydrogen peroxide with the catalase for a longer period of time, then the reaction rate will remain the same.

Procedures
Two 10 mL Pipettes
Catalase
H2O
Eight 50 mL Beakers
2% KMnO4
Three 5mL Pipettes
1M H2SO4
Two 100 mL Beakers
1.5% H2O2
Potato (boiled and nonboiled)

Establishing a Baseline 1. Place 10 mL of 1.5% H2O2 into a clean 50mL beaker. Be sure to use a pipette to get the most accurate reading for the amount of H2O2. 2. Now using a new pipette, that isn't contaminated, place 1 mL of H2O into the 10ml of 1.5% H2O2. 3. Using a new pipette, place H2SO4 into the solution of H2O2 and H2O. Be very careful when using H2SO4; always wear goggles throughout the whole lab to ensure the safety of your eyes. 4. Now mix well using a clean mixing stick. 5. Using a new pipette remove a 5 mL sample of the solution we created. Place the 5 mL solution in a clean beaker. Now place a white sheet of paper behind the beaker. 6. Using a new pipette place 5 mL of KMnO4, one drop at a time, into the 5 mL solution. Swirl the solution until the solution becomes pink or brown. 7. Measure the amount of KMnO4 left in the pipette. Record the amount of KMnO4 left in the pipette and subtract it from the original to get the baseline.

The Uncatalyzed Rate of H2O2 Decomposition 1. Place 15 mL of H2O2 into a 50 mL beaker and store it in room temperature uncovered over night for 24Hr 2. Repeat steps 2-7 from Establishing Baseline
Time Course Determination 1. Place 10 mL of 1.5% H2O2 into a clean 50mL beaker. Be sure to use a pipette to get the most accurate reading for the amount of H2O2. 2. Now using a new pipette, that isn't contaminated, place 1 mL of Catalase into the 10ml of 1.5% H2O2. 3. Swirl Gently for 10 seconds 4. At 10 seconds using a 10ml Pipette add 10mL of 1M H2SO4 5. Take out 5mL sample from solution with 10mL pipette 6.
Use 10mL pipette to slowly add KMnO4 until solution turns persistent pink or brown color 7. Repeat Steps 1-6 for time intervals of 30,60, 120, 180, and 360 seconds

Test of Catalase Activity 1. Cut 1mL (cm3) of potato and macerate it. 2. Prepare 50mL glass beaker with 10mL of H2O2 3. Place 1mL potato into glass beaker and record results 4. Repeat step 2 5. Place boiled 1mL of boiled potato into 50mL beaker of solution

Data KMnO4(mL) | Time(seconds) | | 10s | 30s | 60s | 120s | 180s | 360s | A. Baseline | 3.5 | 3.5 | 3.5 | 3.5 | 3.5 | 3.5 | B. Final Reading | 7.5 | 7.5 | 8 | 8 | 8 | 7.5 | C. Initial Reading | 5 | 5 | 5 | 5 | 5 | 5 | D. Amount of KMnO4 Consumed | 2.5 | 2.5 | 3 | 3 | 3 | 2.5 | E. Amount of H2O2 Used | 1 | 1 | .5 | .5 | .5 | 1 |

Baseline Calculation
Final Reading of Pipette 6.5mL
Initial Reading of Pipette 3mL
Baseline 3.5mL

Rate of reaction: Time | Calculation(Amount decomposed/Time) | Rate | 10s | 1/10 | .1 mL/s | 30s | 1/20 | .05 mL/s | 60s | .5/30 | .0167 mL/s | 120s | .5/60 | .008 mL/s | 180s | .5/60 | .008 mL/s | 360s | 1/180 | .005 mL/s | Average Rate | | .031mL/s
|

Results and Analysis
Based on our chart comparing the time and H2O2 consumption, we cannot tell the story of the data.
As the data is then changed into the rate of reaction based on time, we can tell that the rate of disappearance is slowly decreasing. To get a even clearer idea on what our data looked like, I put the rate of disappearance of H2O2 into a time plot which illustrates rate over time. We can now see the image that the graph is exponentially decreasing at a rapid rate as the time of the mixing increases. The average rate of reaction for our results is .031 mL/s. The data we see is an result of decreasing concentration of substrate. When the catalase is first introduced for 10seconds, the rate of reaction was highest because that was when substrate and enzyme concentration stayed the highest. As the experiment is prolonged, substrate concentration will only decrease as it is limited but the enzymes will continue to catalyze the lower concentration which leads to a lower rate. The amount of H2O2 consumed in the time course determination test never exceeded the baseline because it is expected that the baseline reacts to
completion.
Another part of the experiment was The Test of Catalase Activity which tested potatoes which contained catalace and how they reacted with H2O2. Although our lab group did not get to experiment with this, I expect that the raw potato’s catalase will be in its confirmation state which will be able to catalyze the H2O2 in the beaker. The boiled potato should have no effect on the H2O2 in the beaker of solution as high temperature will cause denaturalization to the catalase.
My conclusion was not supported by my experiment. The rate of reaction was affected by the time because the rate decreased over time because of the decrease in substrate concentration.
Sources of error:
Inaccurate lab equipment leads to skewed results
Contamination of Pipettes can change results slightly
Inaccurate time recording from stopwatch
Inaccurate measuring of liquid in beakers
Catalace temperature as it is left on table for different experiments

Conclusion:
The rate of decomposition of H2O2 genrerally increased with the addition of catalase but as time of interaction increases; the rate is slowly decreases. This is because of the shortage of substrate (H2O2) within the solution,
Future experiments include testing effect of salt concentration and temperature on reaction rate.