Renee Zhu AP Bio 6-7 September 19, 2011 Enzyme Catalysis Lab An enzyme is a type of protein that accelerates

the rate of a chemical reaction while lowering activation energy. Enzymatic activities allow chemicals to react at lower temperatures, and it is possible because enzymes combine with the substrate to form a complex, and then form the product. E + S ES E + P This process allows the enzyme to continue breaking down other substrate molecules again and again. The enzyme, or catalase, weighs about 240,000 daltons and has 4 polypeptide chains and each is made up of more than 500 amino acid residues. Catalase is important to the body because it can help regulate unwanted chemicals that can be harmful, or it can help stimulate important functions in cells. One example of a catalase at work is the reaction between H2O2 and the enzyme to form water and oxygen, in order to prevent harmful amounts of hydrogen peroxide in our systems. Our experiment was to study the catalyzed reactions to determine the rate at which the catalase worked, by measuring the amounts of H2O2 was left after reacting with the catalase after certain periods of time. To determine the initial rate of a reaction, you take the final amount of moles and subtract the initial amount of moles from it, then divide it by the time that passed. This moles product/ second shows how the catalase acts on the substrate and should be constant each time as long as the temperature and pH are constant along with excess substrate. Our hypothesis was that the longer the H2O2 was left with the catalase to react, the more H2O2 would be used. To determine whether or not our hypothesis was correct, the following procedure was used:

Renee Zhu AP Bio 6-7 September 19, 2011 1) Determine the baseline amount of H2O2 by mixing 10 mL of 1.5% H2O2 with 1 mL of water 2) Add 10 mL of H2SO4 to it, and mix well 3) Remove 5 mL and assay by adding KMnO4 until the solution turns into a persistent pink/brown/purple color. The H2SO4 was added to stop the reaction. This happened because the acid denatured the enzyme, which prevented it from reacting with the substrate. After doing this, we determined that the amount of KMnO4 used was 3.1 mL. This meant 3.1 mL was needed to react with all the remaining H2O2, and this was our baseline. Next in the procedure came the enzyme-catalyzed rate of the H2O2 decomposition. 1) Put 10 mL of 1.5% H2O2 in a beaker 2) Add 1 mL of catalase extract (ground up liver w/water) 3) Swirl gently for 10 seconds 4) At 10 seconds, add 10 mL of H2SO4 5) Remove 5 mL and assay with KMnO4 to determine the amount of H2O2 left The titration process (use of KMnO4) was used to determine how much H2O2 was left after the substrate was allowed 10 seconds to react with the catalase. Our class average came out to 2.1 mL We repeated the steps 1-5 but with 30 seconds, 60 seconds, 120 seconds and 180 seconds. Our class averages of KMnO4 used came out as the following: 10 seconds 2.1 mL 30 seconds - .32 mL 60 seconds - .26 mL 120 seconds X (experimental error) 180 seconds - .2 mL

Renee Zhu AP Bio 6-7 September 19, 2011 To determine how much H2O2 had been used in each process, we had to find the amount of baseline H2O2 that had been in the baseline solution. .0031 L of KMnO4 used * .13 moles/L = .00403 moles of KMnO4 used .00403 moles KMnO4 * 2.5 moles H2O2/ mole-KMnO4 = .001 moles of H2O2 This means that .004225 moles of H2O2 were present in the baseline solution. From this we can repeat this process for all of the time trials and then subtract the amounts of moles of the H2O2 from the baseline. At 10 seconds: .0021 L of KMnO4 used * .13 moles/L = .000273 moles of KMnO4 used .000273 moles KMnO4 * 2.5 moles H2O2/ mole-KMnO4 = .0006825 moles H2O2 .001 - .0006825 = .0003175 moles of H2O2 decomposed At 30 seconds: .00032 L of KMnO4 used * .13 moles/L = .0000416 moles of KMnO4 used .0000416 moles KMnO4 * 2.5 moles H2O2/ mole-KMnO4 = .000104 moles H2O2 .001 - .000104 = .000896 moles of H2O2 decomposed At 60 seconds: .00026 L of KMnO4 used * .13 moles/L = .0000338 moles of KMnO4 used .0000338 moles KMnO4 * 2.5 moles H2O2/ mole-KMnO4 = .0000845 moles H2O2 .001 - .0000845 = .0009155 moles of H2O2 decomposed At 180 seconds: .0002 of KMnO4 used * .13 moles/L = .000026 moles of KMnO4 used .000026 moles KMnO4 * 2.5 moles H2O2/ mole-KMnO4 = .000065 moles H2O2 .001- .000065 = .000935 moles H2O2 decomposed

Renee Zhu AP Bio 6-7 September 19, 2011 Time 10 seconds 30 seconds 60 seconds 180 seconds H2O2 decomposed .0003175 .000896 .0009155 .000935

ANALYSIS: From the data, it can be seen that the longer the catalase and the H2O2 were allowed to mix together, the more H2O2 decomposed. It also became clear that after a minute, the data collected after 180 seconds was very close to the data collected after 60 seconds, implying that most of the substrate had already reacted after 60 seconds. Question 3: The Rate is highest between 0-10 seconds because as the reaction continues, there are less molecules remaining to react and the rate slows. Question 4: The rate is the lowest between 60 and 180 because the time is lonver but the difference in amount of chemicals is very small. Question 5: The sulfuric acid caused the catalase to become denatured due to the excess amounts of [H+] ions and this caused the reaction to stop immediately. Question 6: Enzymes work better at optimal temperatures, so lowering the temperature would slow the rate of reaction as less. If temperature decreases, the molecules move slower and therefore react slower. An error that happened to our group was that we read the results wrong when measuring the amount of KMnO4 used. This threw off our molar calculations, so class averages were used instead during our experiment. Overall, the experiment was useful in showing us rate of reactions of enzymes and how it doesnt take long for a substrate to react almost completely with a catalase. The reaction was highest in the first 10 seconds. The sulfuric acid also helped to show how immediately an acid can denature a catalase.