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12 Biology Sol Song, Chelsea Ryu Oct.20/ 2009 Enzymes Introduction: All living organisms require enzymes for efficient and fast metabolic reactions. An enzyme is found to be water-soluble protein, with the exception of hydrophobic lipoprotein in mitochondria. Some enzymes are entirely protein; others consist of two parts: an apoenzyme that is protein, and cofactor or coenzyme that is non-protein. Cofactor is inorganic, but coenzyme is organic. Catalase is a type of enzyme that catalyzes the decomposition of hydrogen peroxide. The reaction is as follows: 2H2O2 --> 2H2O + O2 (the substrate is hydrogen peroxide and the products are the two harmless molecules, water and oxygen). The presence of catalase is crucial to break down the poisonous hydrogen peroxide. If the catalase does not catalyze this harmful chemical, the cells will die. It is mostly found in the peroxisomes and glycoxisomes of plants, such as potatoes, and in the liver and kidney tissues of mammals. In this lab, a potato, liver, and manganese dioxide will be used to study catalase. Is the action of catalase changed under different conditions? If the catalse exists in potato and liver cells, it will react differently when it is reused, and the amount of substrate, particle size, and temperature are changed. Since an enzyme changes its shape, or become denatured, under high temperatures and extremes of pH, it stops function at the certain conditions. Also, if a cell increases in the surface area to volume ratio, it generally speeds up its reaction, for more surface area means more contact with other reactants to initiate the reaction. The higher concentration of substrate is another factor that increases the rate of reaction. However, the reaction speeds up only until all the enzymes are saturated, or completely filled with substrates. Purpose: To compare the action of catalse to a non-protein catalyst under different conditions. Procedure: Materials and Equipment 3% hydrogen peroxide scalpel manganese dioxide 2 stirring rods fresh or frozen liver hotplate potato 250mL beaker, graduated cylinder ice test tubes, test tube rack, test tube tongs fine clean sand lab apron, water soluble marker CAUTION: *Hydrogen peroxide is an aggressive oxidizer that may corrode many materials, even human skin. Do not get any in your mouth. Wash away any spills or splashes with plenty of water. Call your teacher. *Manganese dioxide is also a harmful oxidizer that can cause fire with combustible material. It is harmful to respiratory and nervous systems. In case of contact with skin or eyes, rinse immediately with plenty of water and call your teacher. 1. 2. Put on lab apron and draw your own symbol on each test tube as an indicator. Using a graduated cylinder, measure 4mL of hydrogen peroxide (Measure the bottom of the curvature). Prepare two test tubes and put them in a rack. Add 2mL of hydrogen peroxide into each test tube. Place 0.1g of sand (about a fingernail amount) in one test tube and 0.1g of manganese dioxide to the second tube. Record your observations and 3. Biology 12 Sol Song, Chelsea Ryu Oct.20/ 2009 the rates of reaction after examining the formation of oxygen gas. Discard the materials. 4. Prepare two clean test tubes and repeat step 2. Using a scalpel, cut a small (1 cm3) piece of potato and a slightly larger (2-8cm3) piece of liver, Put the liver in one test tube and the potato in the other. In terms of the rate of reaction, compare the results with those in step 3. Record your observations and the rates of reaction. Discard the potato solution. Reserve the liver solution! Prepare two clean test tubes. Equally divide the liquid portion of the previous test tube in step 3 containing the liver into the two test tubes. Cut the liver from step 3 into two equal sizes and add each portion to each test tube. Then, add a fresh piece (2-8cm 3) of liver to the first and add 1mL of hydrogen peroxide to the second. Record your observations and the rates of reaction. Place a small (2-8cm3) piece of liver in one test tube and a small (1 cm3) piece of potato in the other. Add a pinch of sand to each test tube and crush these materials by using separate stirring rods. Do not grind them violently, which will break the test tubes. Repeat step 2. Record your observations and the rates of reaction, compared to the results in step 3. Put the same size liver in three test tubes. Heat one of them for 10 minutes in a beaker with boiling water (100C). Place the second test tube in a 37C water bath for 5 minutes. Place the third test tube in an ice-water bath (0C) for 5 minutes. Make sure that the test tubes are held with test tube tongs, and do not touch the bottom of the beaker. Remove these three test tubes from the beakers and add 2mL of hydrogen peroxide to each tube. Record your observations and the rates of reaction. 5. 6. 7. Observations: Record your results in a chart similar to the one shown below. Rates of reaction can be designated as follows; 0- no reaction, 1- slow, 2- moderate, 4- very fast. Title: Enzymes Step Experiment A, Catalytic Reactions sand + H2O2 Observations -No bubbling occurs; the sand sinks to the bottom. Rate of Reaction 0 Interpretations Nothing happens because there is no enzyme in the reaction. Biology 12 Sol Song, Chelsea Ryu Oct.20/ 2009 MnO2 + H2O2 B, The effect of an enzyme H2O2 + liver H2O2 + potato -This reaction occurs faster than the first reaction. Small silver bubbles, accumulating on top. -Relatively slower than other active reactions -less number of bubbles -foam is about 0.7cm thick -Large and pink bubbles keep on rising roughly at first time and then stops. -higher bubble-stack than the previous one; foam is about 1cm high Moderate reaction occurs with white small bubbles forming on top. -foam is about 0.6cm high No bubbling occurs. -Large bubbles form rapidly at first time and then stop soon. -foam is about 6cm high -Large bubbles immediately rise and almost brim over the test tube. -foam is more than 10cm high -Thick bubbles form most rapidly. -foam is more than 2cm high -a small amount of big bubbles form very slowly. -foam is about 2cm high -Large and rapid bubbles keep on rising, almost brimming over the tube. -foam is more than10cm high Reaction occurs fast, and large bubbles form. -foam is about 9cm high 2 Manganese oxide is an inorganic enzyme and acts as a catalyst by speeding up the reaction. 3 Liver contains reasonable amount of enzyme (catalase). 2 Lower concentration of catalase in potato causes lower reaction. The substrates had already been reacted, so there is no reaction at all. The enzyme already saturated with the substrates. More rapid bubbles form than the previous one because enzymes are reusable. The crushed liver increases the surface area and the concentration of enzyme. This causes faster, more intense reaction than step . More surface area causes more bubbling than in step . Enzymes denature at high temperature; therefore, a slow reaction occurs. At room temperature which is the optimal temperature (37C), reaction occurs rapidly. Too low temperature causes decrease in kinetic energy and collisions between enzyme and substrate; therefore, a slower reaction occurs. C, Re-using an enzyme used liver + liver+ fresh used H2O2 used liver + H2O2 D, Effect of particle size crushed liver + sand + new H2O2 crushed potato + sand + H2O2 E. Effect of temperature cooked liver + boiling water + H2O2 0 3 4 3.5 1 liver + 37C water + H2O2 4 liver + ice water + H2O2 3 Analysis/Questions: 1. How do you account for the differences in the rate?(0- no reaction, 1- slow, 2moderate, 3- fast, 4-very fast) Biology 12 Sol Song, Chelsea Ryu Oct.20/ 2009 In order to compare the activity rating for all test tubes, the intensity of the oxygen gas bubbles should be examined. I number the results according to the relative size, thickness, speed, and amount of bubbles, and most of all, the height of the foam at the critical point. For instance, in potatoes, the reaction rate of 1 slowly makes 0.5cm-high foam with small bubbles whereas the reaction rate of 3 creates 1cm-high foam with larger bubbles. 2. Can H2O 2 be broken down by catalysts other than those found in living systems? Explain you answer. Hydrogen peroxide can be broken down by catalysts in the presence of any substance that can be oxidized, for H2O2 very reactive and able to naturally decompose over time to produce water and molecular oxygen. Catalysts do not have to be all organic like protein; they are either organic or inorganic. For example, although manganese dioxide is an inorganic chemical that is not found in living organisms, it started the decomposition reaction during the lab. 3. Describe the effect of temperature and particle size on the rate of enzyme action. As the temperature increases, the rate of enzymatic reaction increases because higher temperature causes more effective collisions between a substrate and an enzyme. Especially at the room temperature (37C), an enzyme works most actively; however, too high temperature denatures the protein enzyme by disrupting normal hydrogen bonding and R-group interactions, so with boiling water the enzyme cannot carry out the reaction. At too low temperature, at approximately 0C, the reaction slows down, indicated by forming a fewer small bubbles. As particle size gets smaller, the rate of enzyme action increases because larger surface area of small molecules creates more opportunities to react with the enzyme faster. 4. The body temperature of a dog is approximately 40C. Would your results be different if you had used pieces of dog liver for this investigation? Yes, my results would be slightly different because the enzyme in my body works best at 37C since it is the normal body temperature where enzymes function most actively. Since the optimal temperature for humans is not 40C, the reaction would not occur as spontaneously or vigorously as it starts to denature. Biology 12 Sol Song, Chelsea Ryu Oct.20/ 2009 5. Explain the difference between the rates of reaction with potato as compared to liver. Due to the more instant formation and higher number of large bubbles of oxygen, the rate of reaction with liver is faster than with potato because the liver contains more catalase. Since the liver has a greater metabolism and its toxic by-product, hydrogen peroxide, should be removed, more catalase is required in the liver than in the potato. Conclusion: In this experiment, different reactants in each of the two test tubes were examined under five different conditions. In Step , when manganese dioxide and sand reacted with hydrogen peroxide, no bubbles formed in the solution with the sand, indicating its absence of catalase. However, in the fourth step, both solutions with a potato and a liver created the oxygen gas. Thus, catalase can be both inorganic like manganese dioxide and organic like livers and potatoes. According to the liver solutions thickest foam and highest number of bubbles, catalase in livers was concluded to be the most reactant. Livers contain more catalase since it needs to remove the toxic by-product, resulting from the greatest metabolism. Potatoes, too, require catalase since they perform photosynthesis that causes some toxins but not as much as livers do. As I hypothesized, number of use, particle size, and temperature affected catalase differently. When the substrate hydrogen peroxide reacted with the used enzyme, there was a chemical reaction; however, when the used liver was combined with fresh liver and used substrate, there was no reaction. Since the reactant was already used in the previous step and completely filled, or saturated, the enzyme, the reaction could not be initiated. The enzyme, however, was reusable, for it reacted with the new hydrogen peroxide. In terms of particle size, the ground enzyme with smaller particles had faster rate of reaction that the chunk. Crushing the liver and potato provided more surface areas, or more contacts with hydrogen peroxide. Also, at a certain temperature, 37, the reaction was most active. After the test tube was placed in an ice bath, the formation of bubbles was significantly slower. After it was bathed in hot water at 100, the reaction was faster than the cold enzyme but slower than the enzyme at the room temperature. Therefore, an enzyme can be reused since it does not directly participate in the reaction, but a substrate cannot be reused since it is consumed completely to form a product. An increase in the substrate concentration may not catalyze the reaction after it has saturated the enzyme. Additionally, the small particle size and high temperature generally speed up an enzymatic reaction. Nevertheless, if the temperature exceeds the optimal temperature, the enzyme denatures and the reaction decelerates. One of the possible errors in the lab arose from different individual interpretations of measurement and observation. Without a scale, the measurement of 0.1g of sand and manganese dioxide could not be precise. Also, it was hard to equally cut the liver and potato without using a scale or a ruler. Besides, the liver had to be bigger than the potato in order to easily halve the liver. If the measurements made by an individual were not biased, each reaction rate may have been different. If my liver were smaller than the expected size, my reaction would have been less vigorous, perhaps slower than the reaction of my potato. Next time, I will use measuring equipment or at least try to be consistent with the size for more accurate results. Furthermore, an individuals judgment of the reaction rate may have caused some errors. Whereas I used the thickness of foam primarily as the determining factor, other students may have estimated the number or the size of bubbles. Then, the reaction rate chart may have changed. My hypothesis Biology 12 Sol Song, Chelsea Ryu Oct.20/ 2009 may have been proven wrong if the rate of reaction of potato and liver was found to contain the same number of bubbles. For the effective, fast metabolism, humans need to keep livers healthy and maintain the body temperature. Livers, which contain the most catalase, effectually catalyze the toxins that otherwise poison and kill the cells. Regular exercises and healthy diets (without junk food, trans fat, or saturated fat) will cleanse the liver and prevent from getting sick. Many illnesses generate a raise in temperature, denaturing the catalase enzyme and slowing down the metabolism. ... View Full Document

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