Period5.H

Procedure for Enzymatic Activity Lab Concentration of water and hydrogen peroxide in the solution added to the liver || The amount of enzyme activity in the liver (resulting in the release of oxygen after the breakdown of hydrogen peroxide) ultimately measured in air pressure || Temperature, amount of solution, amount/type of liver, amount of time to break down, and lighting || Introduction: In this experiment we were able to observe enzymes in action. Enzymes, by definition are proteins that catalyze chemical reactions. In essence, they increase the speed of the reaction in order to obtain the final product faster and more efficiently. In this particular lab, we explored the effects of an enzyme named Catalase as it reacts with hydrogen peroxide and water. Catalase can be found in the liver-thus why liver is a crucial component of our experiment. Catalase’s main function is to break down the toxic hydrogen peroxide by hastening its decomposition into oxygen and water or using it to oxidize another organic compound. The hydrogen peroxide is continuously produced by metabolic reactions. Because of this important feature, Catalase is found in the majority of all aerobic cells. Catalase has also been considered to be the basis of aging. It is found that living as oxygen dependent organisms is rather dangerous. Oxgen can be easily converted into other reactive compounds. Carrier molecules are constantly shyttling electron from sito to site. If oxygen runs into one of these carrier molecules, the electron may be transferred to the oxygen molecule. This will, in effect, convert oxygen into potentially harmful compounds, for example superoxide radicals. It could also be converted into hydrogen peroxide, which can attack sulfur atoms and metal ions in proteins. Free iron ion cells may occasionally convert hydrogen peroxide into hydroxyl radicals, which are deadly. They attack and mutilate DNA and may be causing aging. Fortunetly however, catalase fights the dangerous possible effects of living with oxygen. It is used with Superoxide dismutase to convert superoxide radicals into hydrogen peroxide, then catalase converts the hydrogen peroxide into water and oxtgen gas. These two enzymes maintain homeostasis of dangerous productions inside the cell. Though Catalase may or may not be to blame for aging, it is certainly responsible for other very important functions. Each molecule of Catalase has the potential to decompose millions of hydrogen peroxide molecules every second. The reaction that Catalase performs is 2 H2O2 --> 2 H20 + O2. In this lab, we observed whether or not the concentrations of hydrogen peroxide and water affected the Catalase, and if so, how. The purpose was to determine at what percentage of hydrogen peroxide and water the Catalase seemed to have the most affect, and therefore determine which substance would cause Catalase to begin its reaction. To determine our results, we used the reaction previously stated. The reaction will release oxygen as a byproduct and will therefore release more oxygen if more Catalase is being used to break down more hydrogen peroxide. Therefore, a device will be used to measure the oxygen pressure in the flask during the reaction. Overall, this experiment will allow us to further understand enzymes, and the qualitative and quantitative manifestations that arise during a chemical reaction aided by enzymes.
 * Independent Variable || 
 * Dependent Variable(s) || 
 * Controlled Variables || 
 * How will you change your independent variable? (be specific) || We will change the concentration of each solution added by varying the amounts of water in the solution while still controlling the amount of overall solution added to the liver. ||
 * How will you measure your dependent variable? || We will measure the change in air pressure by using the gas pressure probe. If the air pressure increases greatly, this will indicate a release of oxygen that occurs as a result of the breakdown of hydrogen peroxide. ||
 * How will you set up a control? || <span style="font-family: 'Verdana','sans-serif'; font-size: 10pt;">In order to create a control, we will perform all experiments in the same environment, at the same time of day, and maintaining a consistent environment for testing. ||
 * <span style="font-family: 'Verdana','sans-serif'; font-size: 10pt;">Hypothesis: || <span style="font-family: 'Verdana','sans-serif'; font-size: 10pt;">If the concentration of hydrogen peroxide is increased in the added solution, then catalase will perform at a higher rate, releasing more oxygen which will result in an increased air pressure. ||

Materials: 1. Four pieces of 1.00 g. of liver (obtained from local grocery store) 2. Scalpel to cut liver 3. Tongs to handle liver 4. Scale (to mass liver) 5. (Four) 125 mL flasks 6. Tape 7. One marker 8. At least 20 mL of hydrogen peroxide 9. At least 20 mL of water 10. Eyedropper 11. Four 10 mL graduated cylinders 12. One stopper 13. One sensory probe a. To be attached to stopper 14. Timer/clock 15. Computer 16. One pair of goggles per person 17, Lab pro computer program

<span style="font-family: Tahoma,Geneva,sans-serif;"> *NOTE: The Stopper will pop off as the air pressure reaches approximately 125 kPa, when this occurs, simply record the time and pressure when the cap pops off*
 * 1) <span style="font-family: Tahoma,Geneva,sans-serif;">Gather your materials.
 * 2) <span style="font-family: Tahoma,Geneva,sans-serif;">Slice and mass four liver samples of approximately 1.00 gram each.
 * 3) <span style="font-family: Tahoma,Geneva,sans-serif;">Place the liver samples each individually into separate 125 mL flasks.
 * 4) <span style="font-family: Tahoma,Geneva,sans-serif;">Label the four 125 mL flasks as follows:
 * 5) <span style="font-family: Tahoma,Geneva,sans-serif;">Sample 1: 100% water
 * 6) <span style="font-family: Tahoma,Geneva,sans-serif;"> Sample 2: 20% hydrogen peroxide, 80% water
 * 7) <span style="font-family: Tahoma,Geneva,sans-serif;">Sample 3: 80% hydrogen peroxide, 20% water
 * 8) <span style="font-family: Tahoma,Geneva,sans-serif;">Sample 4: 100% hydrogen peroxide
 * 9) <span style="font-family: Tahoma,Geneva,sans-serif;">Using an eyedropper, measure sample each sample as follows into seperate 10 mL graduated cylinders.
 * 10) <span style="font-family: Tahoma,Geneva,sans-serif;">Sample 1: 10 mL of water
 * 11) <span style="font-family: Tahoma,Geneva,sans-serif;">Sample 2: 2 mL of hydrogen peroxide, 8 mL of water
 * 12) <span style="font-family: Tahoma,Geneva,sans-serif;">Sample 3: 8 mL of hydrogen peroxide, 2 mL of water
 * 13) <span style="font-family: Tahoma,Geneva,sans-serif;">Sample 4: 10 mL of hydrogen peroxide
 * 14) <span style="font-family: Tahoma,Geneva,sans-serif;">Hook up the lab pro computer program and components to stopper.
 * 15) <span style="font-family: Tahoma,Geneva,sans-serif;">Pour Sample 1 from the graduated cylinder into the appropriately labeled flask.
 * 16) <span style="font-family: Tahoma,Geneva,sans-serif;">Place stopper firmly on the opening of the flask, making sure to move the nob on the stopper to a parallel position to the table (this keeps oxygen from flowing out of the tube). Do this as quickly as possible.
 * 17) <span style="font-family: Tahoma,Geneva,sans-serif;">Press 'start' on the program to begin collecting data on the lab pro computer program for two minutes.
 * 18) <span style="font-family: Tahoma,Geneva,sans-serif;">At the end of the two minutes stop the computer program and record the starting and ending pressures, as well as your observations of the reaction.
 * 19) <span style="font-family: Tahoma,Geneva,sans-serif;">Repeat steps 6 through 10 with each sample.
 * 20) <span style="font-family: Tahoma,Geneva,sans-serif;">Compare your results for each sample to determine which sample produced the greatest air pressure. This will indicate the greatest amount of oxygen released, and therefore the efficiency of the enzyme catalase, as oxygen is released as it breaks down the hydrogen peroxide.
 * 21) <span style="font-family: Tahoma,Geneva,sans-serif;">Repeat experiment with each sample a minimum of two times to insure accuracy, averaging the pressures together.

Data:

__Hypothesis:__ <span style="font-family: 'Verdana','sans-serif'; font-size: 10pt;">If the concentration of hydrogen peroxide is increased in the added solution, then catalase will perform at a higher rate, releasing more oxygen which will result in an increased air pressure.

__Independent Variable:__ <span style="font-family: 'Verdana','sans-serif'; font-size: 10pt;">Concentration of water and hydrogen peroxide in the solution added to the liver

__Dependent Variable:__ The amount of enzyme activity in the liver (resulting in the release of oxygen after the breakdown of hydrogen peroxide) ultimately measured in air pressure

__Data Table For Observations and Pressure Change in Each Solution__

<span style="font-family: Tahoma,Geneva,sans-serif;">[Graphs on paper]
 * <span style="font-family: Tahoma,Geneva,sans-serif;">Mixture || <span style="font-family: Tahoma,Geneva,sans-serif;">Observations || <span style="font-family: Tahoma,Geneva,sans-serif;">Starting Pressure || <span style="font-family: Tahoma,Geneva,sans-serif;">Final Pressure (after 2 minutes) ||
 * <span style="font-family: Tahoma,Geneva,sans-serif;">20% Hydrogen Peroxide, 80% Water || <span style="font-family: Tahoma,Geneva,sans-serif;">Bubbling a little at the start; mainly occurs around the liver. Pressure on the graph is measured very slowly. || <span style="font-family: Tahoma,Geneva,sans-serif;">99.60 kPa || <span style="font-family: Tahoma,Geneva,sans-serif;">103.97 kPa ||
 * <span style="font-family: Tahoma,Geneva,sans-serif;">80% Hydrogen Peroxide, 20% Water || <span style="font-family: Tahoma,Geneva,sans-serif;">A lot of bubbles across the whole flask. Bubbles build up very fast. || <span style="font-family: Tahoma,Geneva,sans-serif;">108.46 kPa || <span style="font-family: Tahoma,Geneva,sans-serif;">Cap pops off at 127.70 kPa at 49 seconds ||
 * <span style="font-family: Tahoma,Geneva,sans-serif;">100% Hydrogen Peroxide, 0% Water || <span style="font-family: Tahoma,Geneva,sans-serif;">Many bubbles at start, bubbles cover the bottom of the flask completely. Bubbles are coming directly from liver at a rapid rate. || <span style="font-family: Tahoma,Geneva,sans-serif;">111.24 kPa || <span style="font-family: Tahoma,Geneva,sans-serif;">Cap pops off at 124.97 kPa at 46 seconds. ||
 * <span style="font-family: Tahoma,Geneva,sans-serif;">100% Water, 0% Hydrogen Peroxide || <span style="font-family: Tahoma,Geneva,sans-serif;">No bubbles whatsoever. || <span style="font-family: Tahoma,Geneva,sans-serif;">98.47 kPa || <span style="font-family: Tahoma,Geneva,sans-serif;">98.58 kPa ||

Conclusion When examining the results from the liver lab, there seems to be a very apparent correlation between the concentration of hydrogen peroxide and the amount of oxygen (measured by air pressure) present. In our experiment, we observed that the pure water solution did not cause any rise in air pressure. This indicates that the catalase present in the liver was not stimulated to function because it had nothing to break down. Therefore, there was no release of oxygen in the solution. Next, we tested the air pressure of a solution that was 20% hydrogen peroxide and 80% water. This diluted solution did cause a slight increase in air pressure, but it was very gradual, and the rubber stopper stayed for the entire duration of the two minutes. The catalase most likely was able to break down some hydrogen peroxide, but because the solution was highly diluted and there was less hydrogen peroxide present, it was broken down at a slower rate, and oxygen was gradually released. Next, we tested the solution consisting of 20% water and 80% hydrogen peroxide. The data that we collected displayed rapid catalase activity in breaking down hydrogen peroxide, for the air pressure rapidly increased and bubbles formed much more quickly and ferociously than before. The stopper even popped off before a full minute had passed. This indicates that as a higher concentration of hydrogen peroxide is available to break down, the catalase breaks it down much more quickly and efficiently. This concept was also displayed when we tested the pure hydrogen peroxide solution and found that the stopper popped off about halfway through the first minute. In reflection, our hypothesis was correct. We had predicted that if the hydrogen peroxide solution is diluted with water, then the air pressure will be decreased and therefore, less oxygen will be released. This is most likely due to the fact that if hydrogen peroxide is less concentrated, the catalase will break it down at a slower rate, for it cannot break down the water that is present, as well. Catalase must function most rapidly when it is presented with a pure concentration of hydrogen peroxide, for it can break down every aspect of its surrounding solution. This, in effect, would increase the amount of oxygen released as a byproduct as well as increase the speed it is able to do so. Despite our attempts to keep our controls at a constant, our data may have had some flaws. To begin, we had to use only two flasks for four different solutions. After we used the two flasks for the first two experiments, we had to rinse them out with water. Some of this water remained in the flask as we poured in our next solution. This may have diluted the higher concentration solutions, for more water was present on the side of the flask, and slightly changed the concentration that we had been aiming for. In addition, the amount of liver present was one of our controls. However, we measured each liver slice by massing it, which may not have given us a completely accurate amount, for the device only measures to the first 2 decimal places. Furthermore, in order to keep the liver at approximately the same mass, we often had to add an additional piece into the flask. However, we do not know if having two or three separated chunks as opposed to one would alter the data, so we cannot be sure if this affected the data in any unforeseen ways. Lastly, it would have been desirable to keep the time the pressure is measured as a control. However, we were not able to keep this consistent because in the solutions that were less diluted, the air pressure mounted so rapidly that the rubber stopper blew off before the 2 minute increment was over. Therefore, the time each sample was measured was inconsistent. If we were to do this experiment over, we would attempt to obtain four different, dry flasks for each experiment so that there was no excess water inside that might dilute the solutions further than was expected. This way, the concentrations would be kept under a tight control. In addition, perhaps we would measure each liver chunk by its dimensions using a ruler, and later mass them to see if we need to shave off any excess liver. This way, the liver chunks would all be the same approximate shape and all in one piece. Furthermore, we would attempt to keep the time each sample is measured for air pressure consistent by modifying the measuring periods at a shorter time...perhaps 30 seconds or one minute rather than two. We also could have made the more highly concentrated samples less highly concentrated, but this would have taken a bit away from the purpose of our experiment. In conclusion, this lab clearly demonstrated the relationship between concentration of the solution being broken down and the rate of enzyme function. We are now aware that catalase works faster and more efficiently when exposed to a higher concentration of hydrogen peroxide, most likely because there is more surrounding solution to break down.

Analysis Questions:


 * 1) <span style="font-family: Tahoma,Geneva,sans-serif;">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.

<span style="font-family: Tahoma,Geneva,sans-serif;">The results we gathered confirm that the liver in a solution of hydrogen peroxide proved to have the greatest affect on catalase activity. As the concentration of hydrogen peroxide increased, catalase worked at a higher rate. This is because catalase is able to break down hydrogen peroxide. From our results and observations, it can be concluded that the mixture containing 100% hydrogen peroxide concentration showed the most enzymatic activity. The solution that contained a concentration of 100% hydrogen peroxide resulted in the cap popping off at 124 kPa, which shows a rapid increase of oxygen production from the active catalase enzyme. Similarly, the 80% hydrogen peroxide also resulted in the cap popping off at 127 kPa. Using this information, we can see that clearly hydrogen peroxide in large amounts will result in a large increase of catalase activity, and that water may actually aid in these reactions, when in small amounts. As catalase breaks down hydrogen peroxide, oxygen is released. As the concentration of hydrogen peroxide decreased and the concentration of water increased, the enzymatic activity declined and the oxygen pressure stayed at an increasingly similar pressure. The solution containing 100% water showed no effects on catalase and the pressure stayed at around 98. kPa for the duration of the two minutes. This is expected because catalase does not break down water.

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?

One of the problems our group faced was the amount of pressure that built up inside of the flasks. In two of our trials with two different solutions, the cap popped off due to the amount of pressure created during the enzymatic reaction. The solution that contained 100% hydrogen peroxide and the solution that contained 80% hydrogen peroxide and 20% water showed the greatest increase of pressure in the flasks. The error that can be taken from these trials is that the metabolic reactions may not have been finished by the time the cap popped off. The final pressure readings that were recorded may not have been the actual final pressure measurements. One way to avoid this problem is to use a larger flask. In the experiment we used 125 mL flasks. If we were to change the procedure of the experiment we would use larger flasks such as 300 mL. Also, we may have had slightly different amounts of liver. It the liver amounts were different than the reactions would be occuring slightly differently with more or less catalase available. While the general reactions would still be the same, our quantitative data would not have been completely accurate. Another source of error could have been calculating the pressure changes within the flasks. We may have started before or after the reaction actually began to occur, giving the different samples more or less time than all the others. These errors may have negatively effected the controlled environment that we were trying to accomplish.

3. Catalase is located in a cell organelle <span style="font-family: Tahoma,Geneva,sans-serif;">called the peroxisome. What do you think is the function of the perioxisome? Explain your reasoning.

The function of the peroxisome is to get rid of toxic peroxides. <span style="font-family: 'Times New Roman'; font-size: 12pt; mso-ansi-language: EN-US; mso-bidi-language: AR-SA; mso-fareast-font-family: 'MS Mincho'; mso-fareast-language: JA;">A peroxisome is bound by a single membrane and buds of off the endoplasmic reticulum. Peroxisomes do not contain their own genome and replicate by division. Peroxisomes contain the enxymes that will destroy the toxic peroxides inside the cell. A peroxide is a compound that contains two oxygens bound by a single bond. Organic peroxides have the potential to either intentionally or unintentionally begin dangerous polymerization, which is present in things such as explosives. The peroxide that was used in the lab was hydrogen peroxide. Hydrogen peroxide is a naturally produced compound that results from the metabolic reactions during cellular respiration. Catalase is found within a peroxisome so that the cell is able to remove high concentrations of hydrogen peroxide. The cell does this by ingesting a toxin and then converting it into hydropgen peroxide. The body also has peroxisomes that will break down hydrogen peroxide into water and oxygen. The peroxisomes function is to break down peroxides that could potentially damage the cell. The cell can live at lower concentrations of hydrogen peroxide because the compound serves as an antibacterial mechanism.

4. Researchers studying the effects of ecstasy (methylenedioxymethamphetamine or MDMA) on mice, found that ecstasy decreased catalase activity in the liver. They also noticed that ecstasy’s effects on catalase were increased as the temperature of the mouse’s environment increased. Based on this information, can you draw any conclusions about what might be going on inside a person’s body when he/she takes ecstasy at a party? What risks are involved?

The physical activity that someone at a party may do can cause a rise in the body’s temperature. Physical activities such as dancing can cause this temperature change. The increased body temperature of the person can then result in a lessened effect of activity on catalase (if the person has taken ecstasy). If the person consumes alcohol, some of the metabolic processes in the liver can be altered. The reduced activity of catalase in the liver, which breaks down hydrogen peroxide, can result in a more dangerous response to alcohol. With the decreased enzymatic activity of catalase in the liver, the hydrogen peroxide in the consumed alcohol cannot be broken down as quickly and the person will feel the effects of alcohol much quicker and also contain a higher level of alcohol concentration in their body, which can be fatal. The high concentrations of alcohol in the body can lead to brain damage and memory loss and can also lead to liver damage. Overdosing on ecstasy can lead to hallucinations, anxiety, cardiac arrest, and strokes.

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)

For the most effective use of catalase as a cheese-maker, the conditions would have to be optimal for catalase to work. Catalase is used in the dairy industry to remove hydrogen peroxide from the milk. In order for this to work efficiantly, the enzymes would have to be at their optimal levels. Enzymes work at an optimal pH level. The optimal pH of catalase is 7, which is in the middle of the pH scale. The pH level for production would have to be constantly 7. The temperature which catalase works best at is between 36ºC and 39ºC. With these conditions, the production of cheese would be at a high efficiency rate.

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

It is important to make sure that the temperatures never go above the optimal temperature for catalase because the enzyme could become denatured. If an enzyme becomes denatured, it will lose its shape and will not be able to carry out its functions properly. If catalase became denatured, hydrogen peroxide would not be able to be broken down, and none of the functions expected of it would occur. This could have very dangerous repercussions as there would now be a very high amount of hydrogen peroxide in the organism, creating potentially fatal effects.