AP Biology

LAB REPORT: Two Enzyme Catalysis. Group Members: Patrick Perez, Anthony Curatolo, Seung Cha, David Tseng, David Musheyev, and Alexander Coma. Date Submitted: October 28, 2011

Patrick Perez

AP Biology Ms. Rodriguez Brown Patrick Perez

Introduction & Background:

In the lab Two Enzyme Catalysis what we did was observe the conversions of Hydrogen Peroxide into water and oxygen gas with the addition of an enzyme. What we also observed how much oxygen was generated by the addition of enzyme to the catalyzed reaction. Before the lab is completed there is a couple of background information you should be aware of, the most important being that the addition of an enzyme to a reaction will increase the rate of a

reaction. There are also factors that affect the way an enzyme performs and functions during the reaction. PH is one of the factors that affect enzymes. Enzymes usually function at their best and full potential at or near the pH of 7. This is because on the pH scale, 7 is neutral. Meaning it s not too acidic or too basic. Temperature also affects the rate of a reaction. Too much heat can damage enzymes. Other factors that affect enzymes include enzyme concentration, substrate concentration, salinity, activators, and inhibitors. In living organisms almost all of the crucial reactions occur within the cells. Enzymes are highly specific meaning that they only catalyze one kind of reaction. The name for this is enzyme specificity. In these reactions including reactions substrates is the name given to the targeted molecules by the enzymes. In biology enzymes play a crucial role. This is because without enzymes many reactions would not be able to occur within the cells. The reason behind the increased rate of the reaction is that enzymes lower the activation energy of the reaction making the reaction occur at a much faster rate. Enzymes are

responsible for increased reaction rates, formation of temporary enzyme-substrate complexes, and are unaffected by the reaction. Enzymes do a lot, but there are also a few things that they don t do. They don t change the reaction and they don t make reactions that would not occur naturally. From all of this background knowledge what I would like to learn from this lab is how much an addition of an enzyme to a reaction can change a reaction. I want to be able to experience firsthand what enzymes are able to do. My hypothesis is that I think that the addition of an enzyme will enhance the reaction making it occur faster as long as there aren t any facors that can affect the enzyme present. My null hypothesis: The addition of an enzyme will have no effect on the rate of the reaction.

Materials: 2A Materials: o o o Glass Beakers Dropper H2O2

o o o o

Cold Catalase Potato Boiled Catalase Liver

2B Materials: o o o o o o o Burette Glass Beakers H2O H2SO4 KMnO4 Syringe H2O2

2C Materials: o o o o o Glass Beakers KMnO4 Burette H2O2 H2SO4

2D Materials: o o o o o o Stop Watch to record time Catalase Glass Beakers H2O2 KMnO4 Burette

Procedure:

2A: o o o o o o o o Transfer 10mL of H2O2 using a dropper into the beaker. Add 1mL of boiled catalase using a dropper into the glass beaker with H2O2 Mixing the solution Observe reaction. Notice the rate of the reaction with catalase. Transfer 10mL of H2O2 using a dropper into the beaker. Add 1mL of cold catalase using a dropper into the beaker which has H2O2 Mixing solution Observe reaction. Check for difference from the boiled catalase.

Part II of 2A (Liver & Potato) [Living Tissue]

o o o o o o o o 2B:

Transfer 10mL of H2O2 using dropper into a beaker Macerate the bits of potatoes Take the macerated potato and add it into the beaker with H2O2 Observe the reaction. (Should be foaming) Transfer 10mL of H2O2 using a dropper into a beaker. Macerate the liver Take the macerated liver and add it into the beaker with H2O2 Observe the reaction (Should be foaming)

o o o o o o

Add 10mL of H2O2 using a dropper into a beaker. Add 1mL of H2O Add 10mL of H2SO4 into beaker Mix solution well Remove 5mL of solution Place into another beaker

o o o 2C: o o o o o o o o o o o 2D: o o o o o o o

Place the sample under a burette containing KMnO4 Allow the KMnO4 to drop into the solution until it turns pink or brown Record information

Add 15mL H2O2 in a beaker Store it uncovered for 24 hours After 24 hours has passed add 1mL of H2O Add 10mL of H2SO4 into beaker Mix well Remove a 5mL sample using syringe Place into another beaker Now place a piece of white paper over the beaker Place the sample under a burette containing KMnO4 Allow the KMnO4 it drop into sample until it turns pink or brown Record information

180 Seconds is the number of time you should observe the solution Add 10mL of H2O2 to beakers Add 1mL of catalase to the beaker Swirl solution Use a stop watch to time 180 seconds When 180 seconds is up add 10mL of H2SO4 Gather the other group s data. All groups had a different time. My group was 180.

Exercise 2A Questions:

1. A. What is the enzyme in this reaction? Catalase B. What is the substrate in this reaction? H2O2 C. What is the product in this reaction? O2 & H2O D. How could you show that gas evolved is O2? Oxygen is trying to escape; resulting in bubbles. 2. The reaction did not occur because the boiled catalase did not work under boiled conditions. This is because temperatures affect enzymes. 3. The reaction bubbled. The catalase would not be able to work under boiled condtions. Exercise 2B Questions: Base line Calculations Final reading of burette 17mL Initial reading of burette 13.5mL Base Line (Final Initial) 3.6mL KMnO4

Exercise 2C Questions: Uncatalyzed H2O2 decomposition Final reading of burette 13.7 mL 24Hr: 17.2mL Initial reading of burette 10.5 mL 24Hr: 13.7 mL Amount of KMnO4 titrant 3.2 mL 24Hr: 3.5 mL Amount of H2O2 sponatneously decomposed (mL baseline mL KMnO4) 3.5 mL

What percent of the H2O2 spontaneously decomposes in 24 hours? [(mL baseline mL 24hours)/ mL baseline] x 100. -9.375% Exercise 2D Questions:

KMnO4 (mL0) 10 a) Base Line b) Final Reading c) Initial Reading d) Amount KMnO4 Consumed (B minus C) e) Amount H2O2 Used (A minus D) 360 3.4 3.4 8.6 6.1 2.5 .9 30 3.4

Time (seconds) 60 90 3.4 120 3.4 180 3.4

10.2 11.2 8.6 1.6 1.8 10.2 1.0 2.4

11.2 11.5 11.6 11.2 11.5 11.6 .3 3.1 .1 3.3 1drop 3.4

4. Graph the data for enzyme

catalyzed H2O2 decomposition. For this graph you will need to determine the following: a. The independent variable: The time (Use this to label the horizontal (x) axis.) b. The dependent variable: The amount of H2O2 Used (Use this to label the vertical (y) axis.)

Analysis of Results: 1. Data Table below shows the initial rate of the reaction and the rates between each of the time points. The rate of the reaction is the slope of the linear portion of the curve ( y / x).

The Rate of the Reaction Time Intervals (seconds) Initial 0 to Rates 10 0.09 10 to 30 30 to 60 60 to 90 90 to 120 120 to 180 180 to 360

0.06

0.04

0.009444

0.02583

2. The rate is at its highest when it is first added from which is 0 to 10 seconds. This is because the reactions are much quicker in the beginning because of the enzymes. H2O2 and the catalase react when first combined due to its high concentration and they meet at active sites faster. 3. The reaction is at its lowest at the end of the reaction. This is because the reaction has already occurred and is ending. Therefore it is slowing back down to its original state. 4. The effect of the sulfuric acid on catalase function is that it denatures the enzyme. When the pH drops below a range (around 7) the enzyme doesn t function like before. It can cause the enzyme to be damaged. The protein is changed and can no longer interact with the hydrogen peroxide. pH is one of the factors that affects enzymes. 5. The effect of lowering the temperature on the rate of enzyme activity will make the reaction slow down. This is because temperature affects the enzyme. The speed of the reaction will become slower. 6. There should be beakers with mixed solutions. They should all have the same solution. They should all be seen under different conditions. They need to add different enzymes under different conditions include different pH and temperature.

Conclusion:
The purpose of the lab was to show the effect of enzymes (catalase) in reactions. It allows you to see how enzymes can help speed up reactions in biology, and in living organisms. We are able to see that proteins are one of the most important factors in the function of enzymes. We were able to see how certain factors affected the enzymes. Factors such as inhibitors and temperature were present in this lab. My hypothesis was pretty accurate. As for the accuracy of the information and data it was pretty accurate. The only issue was that we didn t have enough time to be able to see the effect of the enzymes. I feel like everything was rushed, but we all managed to see what enzymes can do in a reaction.