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IB Biology SL - Enzyme Activity Lab Performed: Due: Closure: This laboratory involves the use of an enzyme that will react with hydrogen peroxide. The enzyme is catalase and hydrogen peroxide (H2O2) is the substrate. The reaction is as follows: catalase 2H2O2 2H2O + O2 Ingested hydrogen peroxide is a poison, while external use of this substance is not. However, the generation of oxygen gas requires careful handling due to the potential combustion hazard oxygen presents when handled near a: a) heat source - open flame b) spark potential - static discharge Wear safety goggles when pouring hydrogen peroxide. During this lab all data will be based upon in vitro (outside the organism) observations as opposed to in vivo (inside the organism). Also, we are assuming that the amount of gas produced is indicative of enzyme activity. Figure 1. Activation Energy - the energy needed to initiate a reaction Reaction Activation Energy no catalyst (1) 2H2O2 2H20 + O2 EA1 = 72 kcal/mol 2H20 + O2 EA2 = 52 kcal/mol 2H20 + O2 EA3 = 20 kcal/mol iron (Fe) (2) 2H2O2 catalase (3) 2H2O2 1 2 3 EA1 EA2 EA3 - spontaneous reaction Activation Energy (EA) ∆G is negative Hydrogen peroxide is a poisonous by-product of reactions in cells and must Problem: broken down quickly into harmless substances. How does the rate of catalase activity changes over a period of time? Does the amount of catalase present influence how enzyme activity changes over time? Materials: reaction vessels rubber stopper with tubing tweezers 10 ml graduated cylinder 3% hydrogen peroxide 100 ml graduated cylinder crushed beef liver tissue (containing catalase) overflow trough filter paper discs online stopwatch safety goggles Procedure: 1) Set up the overflow trough in the sink and fill it with tap water to a level just below the overflow mark. 2) Submerge the 100 ml graduated cylinder in the trough and invert it so that all the air is displaced with water. Leave the inverted graduated cylinder standing in the trough. 3) Wearing safety goggles, measure exactly 10.0 ml of 3% hydrogen peroxide and carefully pour it into a horizontally positioned reaction vessel. 4) Using the tweezers, soak one filter paper disks in the prepared solution of crushed beef liver tissue. The disc should be well saturated but the excess liquid must be wiped off. Carefully place the soaked disc on the upper surface of the horizontally positioned reaction vessel. Ensure that the liver tissue does not come into contact with the hydrogen peroxide at this time. If bubble are observed, clean the reaction vessel and start again. 5) Place the stopper with rubber tubing onto the reaction vessel so that the stopper makes an airtight seal. Place the reaction vessel beside the overflow trough and extend the tubing so that it is placed into the inverted 100 ml graduated cylinder. 6) Revolve the reaction vessel to allow the hydrogen peroxide to come into contact with the disc. Accurately measure the volume of O2 gas produced every 10 seconds for a period of 2 minutes. Be sure that the disc remains in contact with the hydrogen peroxide throughout the 2 minute period. 7) Empty the contents of the reaction vessel into the waste container and thoroughly clean the reaction vessel with soap and water. 8) Repeat steps 2-7 four more times with one disc of liver tissue. 9) Repeat steps 2-8 with three discs of liver and again for five discs of liver NB: Be sure to wash glassware thoroughly between trials. Using the group data, each individual must clearly illustrate the rate of enzyme activity during each 10 second interval, through the 2 minute time period, for the 3 different amounts of liver tissue. You will be evaluated for Analysis, Evaluation and Communication