Period5.A

Jamie Horrigan, Ryan Page, and Sam Chirco Mrs. Odierna March 27, 2010
 * Liver and Hydrogen Peroxide Enzymatic Activity Lab**: Change in Enzymatic Activity with Different Amounts of Liver

Introduction: When beginning this lab, we already had an extensive understanding of enzyme structure and function. An enzyme is a protein that catalyzes reactions by lowering the activation energy needed for a reaction to occur. Enzymes are substrate specific, meaning that they only can only bind to certain substrate molecules. In this lab, liver is used, which contains the enzyme catalase, and hydrogen peroxide(H2O2), which is the substrate in this case. The objective in this experiment is to find out if the amount of liver affects the rate and extent of the reaction. The hypothesis states that i f we increase the amount of liver which contains more of the enzyme catalase, then a greater amount of catalase will produce a larger reaction as shown by bubble produced.  This is because there is more surface area for reactions to occur on a larger sample, and a greater amount of liver contains more catalase, so more of the hydrogen peroxide should be broken down in a larger sample of liver compared to a smaller sample. The enzyme catalase breaks down H2O2 into its byproducts: oxygen and water. Enzyme activity is determined by measuring the amount of bubbles produced when the liver is placed in the hydrogen peroxide solution. -Same liver source -same amount of hydrogen peroxide(100mL) -same 650mL beakers -same thermometer -same pipets || .5g 1g 1.5g 2g || **Materials:** -5, 650mL beakers -5g of cow liver -balance -500mL hydrogen peroxide -ruler -stopwatch -graduated cylinder[greater than 100mL]
 * **Independent Variable** || Amount of Liver (g) ||
 * **Dependent Variable[s]** || Amount of enzyme activity (bubbles) (mm) ||
 * **Controlled Variables** || -Temperature
 * **How will you change your independent variable? (be specific)** || We will add different amounts of liver in each beaker. The amounts are as follows.
 * **How will you measure your dependent variable?** || We will measure the dependent variable by measuring the bubbles produced, because bubbles show the enzymatic activity. The more bubbles there are, the more enzymatic activity is taking place. We will put 100mL of hydrogen peroxide in the beaker, then measure the increase in volume of the bubbles. ||
 * **How will you set up a control?** || Our controlled experiment will be a beaker filled with hydrogen peroxide and it will not contain any liver. Then, the hydrogen peroxide solution will be observed to make sure there is no enzymatic activity. ||
 * **Hypothesis:** || If we increase the amount of liver which contains more of the enzyme catalase, then a greater amount of catalase will produce a larger reaction as shown by bubble produced. ||


 * Procedure** :
 * 1) Gather your materials and arrange them on a lab station.
 * 2) Label the five, 650mL beakers as the control, .5g, 1g, 1.5g, and 2g.
 * 3) To set up the control, add 100mL of hydrogen peroxide to the beaker.
 * 4) Wait five seconds, then record any observations regarding a possible reaction.
 * 5) Cut the liver into several tiny pieces.
 * 6) Place a 650mL beaker on a balance, and then zero it in.
 * 7) Add small pieces of liver to the beaker located on the balance, until the mass is approximately .5 grams.
 * 8) Repeat steps 5-6 until there are four beakers containing .5g, 1g, 1.5g, and 2g of liver.
 * 9) Add 100mL of hydrogen peroxide to each of the beakers, one at a time.
 * 10) Wait precisely five seconds, and then measure the extent of the enzymatic activity as shown in the next step.
 * 11) Measure the volume of the contents of the beaker by looking at the measurements on the side of the beaker.
 * 12) If bubbles appear from oxygen being released as the reaction, measure the amount of bubbles by finding the new volume.
 * 13) Find the volume by looking at the side of the beaker
 * 14) Then subtract the original volume from the new volume.
 * 15) The difference between the two volumes represents the volume of the bubbles, which actually measures how much oxygen is released from the reaction.
 * 16) Repeat steps 8-12 for each of the beakers, and record all observations and measurements.

**Analysis Questions:** 1. What do your results tell you about how your variable affected catalase activity? Include specific data from your experiment to help you answer this question. Our variable was the amount of liver which we added more of in each beaker by .5 g increments. The results told us that the more liver there is, the more enzymatic activity occurs. When we added more liver, which acts like catalase, there were more bubbles which signaled enzymatic activity. It is clear that the amount of bubbles(mm) steadily increased as the amount of liver(g) increased.
 * ~ Amount of Liver (g) ||~ Bubbles Height (mm) ||
 * 0 g || 0 mm ||
 * .5 g || 16 mm ||
 * 1.0 g || 20 mm ||
 * 1.5 g || 39 mm ||
 * 2.0 g || 42 mm ||
 * 0 g || 0 mm ||
 * .5 g || 16 mm ||
 * 1.0 g || 20 mm ||
 * 1.5 g || 39 mm ||
 * 2.0 g || 42 mm ||

2. What were some sources of error and how might they have affected your data? What changes would you make to your procedure –or—what changes did your peer editor suggest that you make? ====Fortunately, our data came out linear with a correlation of 100%. That is not to say that there was no room for mistakes in this lab. One source of error could have occurred if we did not measure the bubbles at exactly five seconds after the liver was placed in the hydrogen peroxide. Another source of error could be measuring the highest or lowest points of the bubbles from the reaction because the bubbles were relatively uneven in height all around. This could have caused for different results. Perhaps in the procedure we should add to always measure the same part of the bubbles, such as the peak or the lowest point.==== 

3. Catalase is located in a cell organelle called the peroxisome. What do you think is the function of the perioxisome? Explain your reasoning.

=
The peroxisome probably functions in metabolic processes and the breakdown of hydrogen peroxide because it contains catalase which is an enzyme that breaks down H2O2, and the “perox” in peroxisome is related to hydrogen peroxide. Peroxisomes are probably found in the liver because the liver is known for containing the enzyme catalase. Since peroxisomes detoxify alcohol and the liver does as well, it is evident that peroxisomes are the cause for detoxification of toxins in the liver.=====

4. Catalase is an enzyme that initiates the breakdown of hydrogen peroxide in the liver. When an individual consumes the drug, ecstasy, there is decreased catalase activity in the liver, so the individual cannot metabolize alcohol normally and therefore becomes drunk quicker. Since catalase cannot act properly when under the influence of ecstasy, levels of toxic hydrogen peroxide build up in the body, which is poisonous. In addition, ecstasy’s effects on catalase are increased as the environment’s temperature increases, so it is very dangerous for a person at a party to take ecstasy. The individual is probably dancing at a party, that is already fairly warm, so without catalase acting on the hydrogen peroxide a person can become hypothermic from overheating. Ecstasy also affects the brain stem by killing irreplaceable nigral cells, causing a person to contract Parkinson’s disease when 240,000 of the cells are killed. Therefore, ecstasy can lead to hyperthermia, liver failure, and Parkinson’s disease.

5. Catalase is used commercially whenever hydrogen peroxide is used as a germicide. For example, it breaks down the H2O2 that was used to pasteurize milk prior to cheese-making (Chu //et al//. 1975).

a. As a cheese-maker, what conditions would you want to have in your cheese factory to ensure that catalase was working best? (use the class findings to help you answer this question)  You would want to have a constant temperature (constant enough not to change temperature in enzymes) and that the germicide that was used is actually H2O2. If not, those eating the cheese will get multiple oxygen bubbles. Also, the catalase would be present but not breaking anything down. You also want to have enough catalase in the cheese factory, so that all of the harmful toxins such as hydrogen peroxide are broken down in the cheese, which limits carbon dioxide bubbles in the blood.

b. Why would you need to make sure that the temperature of the catalase never went above a certain temperature?

Catalase is an enzyme. Enzymes are very finicky when it comes to temperature and pH levels. The catalase will not operate if it is not at its preferred temperature if the temperature of the catalase went too high, it would stop working and the cheese would not achieve its best quality or any quality for that matter. Once a really high temperature is achieved, the enzymes will denature because the the beta-pleated sheets, and alpha helices will unravel, which makes the protein nonfunctional.


 * Conclusion:**

In this experiment, the effect of the amount of liver added to a 100mL hydrogen peroxide mixture was tested with four different samples of liver. For the control, 100mL of hydrogen peroxide was added to an empty beaker, but nothing occurred because there is no catalase to break down hydrogen peroxide in the beaker. Catalase is an enzyme found in the liver that is needed to break down hydrogen peroxide, so it is not toxic to the body. In the second beaker, .5g of liver is added along with 100mL of hydrogen peroxide. The hydrogen peroxide immediately starts to bubble up, from the catalase in the liver breaking hydrogen peroxide into two molecules of water, and one molecule of oxygen. After five seconds, there were sixteen millimeters of bubbles, but the reaction was still going. The foam almost bubbled over the top of the beaker, and it kept bubbling until all of the hydrogen peroxide had been converted into water and oxygen. The beaker also became moderately hot, which proves that heat is a waste product of the reaction. In the third beaker with 1.0g of liver, 20mm of bubbles were produced, showing that the more liver that is in the beaker, the faster the reaction will take place. The fourth beaker contained 1.5g of liver, and produced a 39mm reaction, followed by the fifth beaker that contained 2.0g of liver, and produced 42mm of bubbles. The results show that as the amount of liver increases, the rate of the reaction increases too. This is because a greater mass of liver contains more of the enzyme catalase than a smaller piece of liver, so more catalase can break down hydrogen peroxide at a quicker rate than a smaller number of catalase enzymes. The evidence that the rate of the hydrogen peroxide breaking down corresponds directly with the amount of liver is shown by the following equation: Y= -38.66x4 + 152x3 – 184.33x2 + 91x. “X” represents the amount of liver in grams, and “y” represents the height of the bubble produced in millimeters. In addition, R2 = 1, meaning that the amount of liver corresponds exactly with the reaction produced. The hypothesis we tested was “ If we increase the amount of liver in the liver and hydrogen peroxide mixture, then there will be more oxygen produced by the chemical reaction. ” Our hypothesis was absolutely true. The .5g liver chunk produced few bubbles compared to the other samples. The 2g liver chunk produced enough oxygen bubbles to overflow in a matter of seconds. The results were phenomenally accurate with R 2 equaling 1. Despite our success, there was much room for error. We found ourselves having a tough time measuring the bubbles five seconds after the H2O2 was added. It usually took seven to ten seconds, in which point, the height probably changed a bit. We also could have exposed the H2O2 to an excess amount of air prior to adding it to the liver. The third mistake we could have made was that we could have contaminated the liver by accidently touching it or breathing on it. Despite the inaccuracies, we were fairly successful in this experiment.