Elizabeth van Dijk 02 18 14 Bio 183 006B Properties of Enzymes and the Effects of the Metal Copper Sulfate CuSO on Catalase Abstract In this lab we explored the properties and role of enzymes in biological systems This was accomplished by observing the addition of hydrogen peroxide to water a piece of potato and the enzyme 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 the concentration of metal copper sulfate increases the enzyme activity decreases Thus the addition of 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 CuSO 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 of Biology 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 H O and 5 0 mL 3 H O the second 1 4 X 1 4 potato cube and 5 0 mL H O and the third 1 mL Catalase and 5 0 mL H O For each tube observe and record the data immediately after the addition of H O Then 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 tubes A and B In test tube A add 1 mL Catalase and 2 drops of CuSO then swirl the tube 5 0 mL 3 H O is then added In test tube B make a solution of 1 mL Catalase and 5 drops of CuSO swirl the solution then add 5 0 3 H O Observe the tubes for 3 5 minutes and record the data immediately after the addition of H O in each tube The 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 H O to tube B Then 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 H O respectively Immediately after the H O was added cover the tube with foil invert and add the solution to a cuvette and place inside the spectrophotometer Then measure the absorbance 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 a solution of 6 0 mL dH O 100 L guaiacol 2 drops of CuSO and 150 L H O It is then inverted poured in a cuvette and put into the spectrophotometer and set as the blank reading Tube A is then made as a solution of 1 0 mL catalase 5 0 mL dH O 100 L