Jessica Hughes Ap Biology.

1st period 9/18/11

AP Biology Lab 2 Enzyme Catalysis

Introduction An enzyme is a protein, one of the four macromolecule groups. They are created by cells to act as a catalyst in a reaction, to lower the activation energy and speed up the reaction rate. One type of enzyme, the one tested in this experiment, is formed in the human body, catalase. Catalase breaks down hydrogen peroxide in Eukaryotic cells before it has time to damage the cell. In an enzyme/catalyst reaction, the substrate attaches to the active site of an enzyme. Every active site is different and specific for the substrate it will attach to, it is called induced fit, which requires ATP. This reaction is reversible. 2H202 (catalase) 2H20 + O2 Enzyme reactions can be affected by many factors including concentrations of salt, temperature, pH, and inhibitors. Any of these things can affect the enzyme because it will denature it, which will change the function of the enzyme and the substrate can no longer attach. A chemical reaction will occur over a period of time, so the longer the enzyme if allowed to react with the substrate, the more the reactants will break down. Even if the catalase is not added to the hydrogen peroxide in this experiment, it will still decompose spontaneously just much slower. Hypothesis If an enzyme (catalase) is added to hydrogen peroxide then there should be a significant reaction present. If the temperature of an enzyme is increased then it will denature and there will be no reaction. If Eukaryotic cells naturally produce the enzyme catalase, then there should be a reaction when hydrogen peroxide is added to a potato. If sulfuric acid is added to the reaction then it will denature the enzyme and the reaction will come to a halt. If a reaction with an enzyme is given more time then the decomposition of hydrogen peroxide should speed up significa Materials

A. B. C. D. Hydrogen peroxide 1.5% 60 ml Catalase working solution 6 ml Sulfuric acid 1M 60 ml Potassium permanganate 2% 1 pipet 1 20 ml syringe 6 50 ml beakers Hydrogen Peroxide 1.5% 10 ml Catalase working solution 1 ml Sulfuric acid 1 ml Potassium permanganate 2% 10 ml 1 pipet 1 ml 1 syringe 20 ml 1 titration syringe 3 50 ml beakers Hydrogen Peroxide, 1.5% 10 ml Catalase working solution 1 ml Sulfuric acid 1 ml Potassium permanganate 2% ml 1 pipet 1ml 1 syringe 20 ml 1 titration syringe 2 50 ml beakers Hydrogen peroxide 1.5% 30 ml Catalase working solution 1 ml 1 ml boiled catalase working solution 1 20 ml syringe 1 1 ml pipet 1 glass rod 1 knife or scapel potato 2 50 ml beakers

Procedures

A. Testing Enzyme Activity 1. Label the syringes that will be used throughout the experiment 2. Using a syringe add 10 ml of hydrogen peroxide to a 50 ml beaker 3. Using the pipet, add 1 ml of catalase solution to the beaker 4. Swirl the contents and record observations Effect of Extreme Temperature on Enzyme Activity 1. Using the syringe, add 10 ml of hydrogen peroxide to a beaker 2. Add 1 ml of boiled catalase to the solution 3. Mix contents and record observations Presence of Catalase in Living Tissue 1. Obtain a sliver of potato using a scalpel 2. Place the tissue in a 50 ml beaker 3. Add 10 ml of hydrogen peroxide, observe the reaction B. Establishing a Baseline Determine the Amount of Hydrogen Peroxide in a 1.5% solution 1. Dispense 10 ml of hydrogen peroxide into a 50 ml beaker 2. Add 1 ml of distilled water 3. Using a syringe add 10 ml of sulfuric acid to the beaker 4. Transfer 10 ml of the mixture into a new beaker 5. Fill titration syringe with 10 ml of potassium permanganate 6. Titrate the solution until it turns pink or brown, record final volume in the syringe C. Rate of Hydrogen Peroxide Spontaneous Decomposition 1. Let 25 ml of hydrogen peroxide sit at room temperature for 24 hours 2. Dispense 10 ml of hydrogen peroxide into a new beaker 3. Add 1 ml of distilled water 4. Add 10 ml of sulfuric acid to the beaker 5. Transfer 10 ml of the solution to a new beaker 6. Fill titration syringe with 10 ml of potassium permanganate. 7. Titrate the solution until it turns pink or brown. D. Rate of Hydrogen Peroxide Decomposition by Enzyme Catalysis 1. Dispense 10 ml of hydrogen peroxide into a 50 ml beaker 2. Using the pipet, add 1 ml of catalase to the beaker for 10 seconds 3. Add 10 ml of sulfuric acid to the solution 4. Transfer 10 ml of the mixture to a new beaker 5. Fill the titration syringe with 10 ml of potassium permanganate 6. Titrate the solution until it turns pink or brown 7. Repeat the procedure for 30, 60, 120, and 180 seconds

Data Table 2.1: Enzyme Activity Activity Enzyme activity Effect of Extreme Temperature Presence of Catalase in Living Table 2.2: Establishing a Baseline Titration Syringe Initial reading Final reading Baseline (Initial Final) Table 2.3: Hydrogen Peroxide Spontaneous Decomposition Initial Reading Final Reading Volume Used After 24 Hours Amount of hydrogen peroxide Spontaneously Decomposed (baseline volume used after 24 hours) % of hydrogen peroxide spontaneously decomposed (amount hydrogen peroxide spontaneously decomposed/baseline) x 100 10 ml 6 ml 4 ml 10 ml 3 ml 7 ml -3 ml 75% Observations Bubbling, fizzing, molecules are moving No reaction Thick layer of foam, potato floats, bubbling on the bottom

Table 2.4: Hydrogen Peroxide Decomposition by Enzyme Catalysis Time (Seconds) 10 30 60 120 Baseline 4ml 4ml 4ml 4ml Volume Initial 10ml 10ml 10ml 10ml Volume Final 6.5 ml 6ml 7ml 9ml Volume Amount KMnO4 3.5ml 4ml 3ml 1ml Used (Initial-final) Amount hydrogen peroxide used .5ml 0ml 1ml 3ml (baseline KmnO4 used)

180 4ml 10ml 9.9ml .1ml

3.9ml

Graph 2.1: Time vs. Hydrogen peroxide decomposed by catalase

1. What is the function of enzymes in a living system? It is a protein that acts a catalyst and speeds up reactions that take place in the body (ex. Metabolism) and reduces activation energy. 2. Different enzymes work better under different conditions. Where in a human body might it be beneficial to have enzymes work well in very acidic environments? The stomach 3. There is a large amount of catalase found in a human liver. Does the liver break down more hydrogen peroxide in the summer or winter? Explain your answer. Since the body has a constant temperature during the summer and winter, the rate of hydrogen peroxide broken down should remain the same all year. 4. Many enzymes end with ase. Come up with your own enzyme, then name and explain what this enzyme does. Draw the enzyme and substrate in the space provided below along with the enzyme substrate complex. Hairlase, this enzyme would be created near the scalp of a person, also known as a hair follicle. Could speed up the production of hair growth, only on the scalp however.
Substrate Enzyme

5. Recent advances have allowed humans to mass-produce certain enzymes. Research one such enzyme and explain how this enzyme has been used to benefit society. Tag polymerase, was found in bacteria that lived near hydrothermal vents, but now E. coli has been created to produce it. It is important because it helps in genetic sequencing and discovering other diseases. 6. Amylase is an enzyme that aids in the digestion of starches and has ideal temperature range of 35-40C (approximately human body temperature. Predict what would happen to the rate of amylase activity at 25C. The enzyme would denature when its ideal temperature range is lowered, therefore the rate of amylase activity would decrease significantly if it were even to work at all. 7. In part A of the experiment you observed the formation of gas bubbles in the reaction mixture. What gas do you think was forming? What test do you think you could employ to see if you were correct? Since the hydrogen peroxide was being broken down, oxygen gas was most likely being formed in the reaction. You could determine if there was oxygen being produced by placing a glowing splint of wood near it and carefully determining if there is in ignition. 8. Identify different steps in the procedure that may lead to erroneous results When doing the titrations, it is very possible that the syringe levels were misread. Time trials were not exact and could have allowed the reaction to go longer than it should have, messing up results. 9. When you added the catalase to hydrogen peroxide, you observed a strong reaction in the mixture. Do you think this reaction will continue indefinitely? Why or why not? No, because after a certain amount of time all of the hydrogen peroxide will have been decomposed and there will be no more reaction. 10. In part D, you investigated the rate of hydrogen peroxide decomposition over time. What purpose did each of the following serve in he experiment? Catalase Was the enzyme that sped up the breaking down of the hydrogen peroxide Hydrogen Peroxide the solution we were experimenting on and taking data from its decomposition rate Sulferic acid used to stop the reaction of catalase and hydrogen peroxide Potassium permanganate Was the color indicator to determine the pH level of the reaction.

11. Before performing part D of the experiment, why was it necessary to establish a baseline? Was needed to base the results on 12. You may have observed the reaction of naturally-occurring catalase in tissue from either liver or potato. Design an experiment to determine if the amount of catalase varies from tissue to tissue (e.g. 200 g of liver compared to 200 g of potato). Test the amount of enzyme reaction by using a piece of the outside layer of a potato, and the very center to determine if more catalase is produced in different areas of a potato. 13. Of the thousands of enzymes known, there is a family of enzymes called proteases that catalyze a reaction breaking down proteins. What do you think would happen if you added a protease to your sample of catalase before proceeding with your experiment. It would likely denature the catalase since an enzyme is a protein. Error Analysis Slight error could have occurring because of the temperature of the enzyme not being continuously kept chilled, possible it could have denatured slightly. It is obvious we had serious error in either a misreading confusion for our amount of Hydrogen peroxide spontaneously decomposed (table 2.3), it is impossible to have a negative volume however that is what our calculation resulted in. We also had error between the 1st and second time trials (table 2.4 and graph 2.1), it should have been a continuous positive curve, but ours dipped down at one point. Conclusion In part A we can accept the hypothesis because we did have a significant reaction when catalase was added to hydrogen peroxide, there was no reaction when temperature was tampered with, and there was significant reaction with the potato making us believe there was in fact naturally occurring enzyme in the tissue. All of these observations we recorded in table 2.1 The sulfuric acid halted the reaction, so we were able to determine a baseline that would be needed for the remainder of the experiment (table 2.2) Again we can accept our hypothesis In section C we had error with our amount of hydrogen peroxide, we should have been more careful with readings and noticed our error earlier so we could have fixed it and redone the section before it was too late. Table 2.3 shows this error.

In section D we could again accept the hypothesis to an extent; it is likely we had a systematical error when our second time trial went in the opposite direction we predicted. There are numerous ways this could have happened, but most likely was due to misreading or too much/too little time. For the most part however, our results were accurate and what were looking for, see table 2.4 and graph 2.1.