Elizabeth van Dijk02/18/14Bio 183-006BProperties of Enzymes and the Effects of the Metal Copper Sulfate (CuSO₄) on CatalaseAbstract: In this lab we explored the properties and role of enzymes in biological systems. This wasaccomplished by observing the addition of hydrogen peroxide to water, a piece of potato, and theenzyme catalase. Also, we observed and recorded data of catalase changes under the addition of metal copper sulfate to the environment. In order to fully understand the effects of environment factors on catalase, we monitored the activity of different effects using a spectrophotometer. First, we did an experiment to measure the rate of a catalase reaction. Second, we created our own experiment to measure the rate of the catalase reaction under different environment effects. The experiment also tested the effect of copper sulfate on catalase. The results showed that as theconcentration of metal copper sulfate increases, the enzyme activity decreases. Thus, the additionof heavy metals, such as copper sulfate, denatures catalase as the concentration increases. Introduction:This lab focused on the study of catalase which is a specific enzyme. An enzyme is a biological catalyst which speeds up the rate of a chemical reaction. Enzymes can increase the rate of reaction by lowering the activation energy of the reactions (Department of Biology NCSU, 2014). Without catalysts, metabolic processes would happen to slowly for cells to function. Enzymes act upon substrates, which in this lab was hydrogen peroxide. They also produce products, in the case of catalase are oxygen and water. Substrates bind to active sites on the enzyme forming a complex that is held together by non-covalent bonds (Department of Biology NCSU, 2014). Once it binds to the active site, substrates may undergo changes before they are released into the solution as a product. Enzymes do not undergo changes and can freely engage with another substrate. This function allows enzymes to be recycled. There are certain instances in which a substance can bind to an active site and interfere with the enzyme activity. These substances, called inhibitors, can bind temporarily or irreversibly, which would shut down the enzyme permanently (Department of Biology NCSU,2014). Also, enzymes obey the concept of specificity in which they often bind to a specific molecule or region on molecules. In the case of catalase, it specifically binds with hydrogen peroxide. Catalase is used to speed up the breakdown of hydrogen peroxide into water and oxygen. Because hydrogen peroxide builds up quickly, catalase is a vital component to keep concentrations low. Besides binding to specific molecules, enzymes also have specificity towards certain environmental conditions: pH, temperature, concentrations of salt and other molecules. These conditions can affect the activity of an enzyme and could even alter it physically causing denaturation. If an enzyme is denatured, it can undergo a conformational change to the point where it can no longer function as an enzyme (Department of Biology NCSU, 2014). This experiment focused on the effect of copper sulfate (CuSO₄) on the reaction of catalase. Heavy metals can denature enzymes at certain concentrations and even low concentrations can affect the activity of an enzyme. In a study by Sukhdev Singh and P. Sivalingam, it was observed that copper toxicity has a strong effect on catalase activity. While studying the effects of metal pollution in fish, they were able to determine that copper was the strongest inhibitor of catalase when compared to other heavy metals. This in vitro study showed that high concentrations were required to inhibit catalase activity, but in areas of high pollution, organisms can obtain the same amount of metals (Singh and Sivalingam 1982). To measure the reaction of an enzyme, the decrease in substrate or the amount of product form needs to be monitored. Because it is unclear to define between the amount of hydrogen peroxide and water, an indicator must be added. In this experiment, guaiacol was used because it changes color with detection of small amounts of oxygen gas. The rate of reaction can be measured using a spectrophotometer that quantifies the color change over time (Department ofBiology NCSU, 2014). When the solution gains color, it absorbs more light from the spectrophotometer. Methods:The first part of the lab is to observe the effects of catalase as an enzyme. To do so, test the effects of hydrogen peroxide on water, a piece of potato, and a solution of catalase. Label three test tubes in preparation of the experiment. In the first tube, add 1 mL H₂O and 5.0 mL 3% H₂O , the second 1/4 X 1/4" potato cube and 5.0 mL ₂ H₂O , and the third 1 mL Catalase and ₂5.0 mL H₂O . For each tube, observe and record the data immediately after the addition of ₂H₂O . ₂Then complete a test to observe the effects of an environmental factor on catalase. Specifically, test the effects of metal copper sulfate (CuSO ). Begin by labeling two test tubes₄A and B. In test tube A add 1 mL Catalase and 2 drops of CuSO then swirl the tube. 5.0 mL ₄3% H₂O is then added. In test tube B make a solution of 1 mL Catalase and 5 drops of ₂CuSO , swirl the solution, then add 5.0 3% ₄ H₂O . Observe the tubes for 3-5 minutes and ₂record the data immediately after the addition of H₂O in each tube. ₂The second part of the experiment is completed using a spectrophotometer to monitor the rate of the catalase reaction. First, gather two test tubes and label them B (blank) and 1. Then add 6.0 mL dH O, 100 L guaiacol, and 150 L 0.1% ₂ μ μ H₂O to tube B. ₂Then cover the tube with foil and invert it. A cuvette is then filled up to the fill line with the solution from tube B and used in the spectrophotometer as the blank value. Tube 1 is then filled with 1.0 mL catalase, 5.0 mL dH O, 100 L guaiacol, and 150 L 0.1% ₂ μ μ H₂O , ₂respectively. Immediately after the H₂O was added, cover the tube with foil, invert, and add₂the solution to a cuvette and place inside the spectrophotometer. Then measure theabsorbance values of Tube 1 each minute for 5 minutes. The absorbance values are recorded at 470 nm. The last part of the lab is to create an experiment testing one of the effects of environmental change on catalase. Again, test the effect of copper sulfate. Four test tubes are gathered and labeled Blank A, Blank B, Tube A, and Tube B. Blank A is