Testing for Catalase Enzyme Activity with Filter Paper Disks Introduction Hydrogen peroxide (H 2 O 2 ) is a poisonous byproduct of metabolism that can damage cells if it is not removed. Catalase is an enzyme that speeds up the breakdown of hydrogen peroxide into water (H 2 O) and oxygen gas (O 2 ). Catalase enzyme 2H 2 0 2 2H 2 O + O 2 A CATALYST is a substance that increases the rate of the reaction without being used up in the process. CATALASE is an enzyme, a biological (organic) catalyst. Hydrogen peroxide is the substrate for catalase. Methods The assay system used in this lab consists of a filter paper disc which is coated with the enzyme and then dropped into a vial (microtiter plate*) of substrate (hydrogen peroxide). As the enzyme breaks down the hydrogen peroxide into water and oxygen gas, the bubbles of oxygen collect underneath the filter disc and make it rise to the surface of the hydrogen peroxide. The time it takes for the filter disc to rise is an indication of the rate of enzyme activity. You will be doing 5 repetitions for each experiment. Rate: Rate of enzyme activity = distance (depth of hydrogen peroxide in mm)/time (in sec). We will assume that each filter is coated with the same amount of catalase (except in the investigation of the effect of enzyme concentration on enzyme activity). Enzyme: The catalase enzyme has been prepared for you as follows: 50g of peeled potato was mixed with 50 ml cold water and crushed ice and homogenized in a blender for 30 seconds. This extract was filtered through cheesecloth and cold water was added to a total volume of 100 ml. Extract concentration is arbitrarily set at 100 units/ml. ENZYME SHOULD BE KEPT ON ICE AT ALL TIMES!! Question #1: What is the effect of enzyme concentration on rate of reaction? 1. Make a large number of filter paper discs using a coffee filter and a hole punch. 2. Measure the depth of one sample vial on the microtiter plate. (This will be needed in order to determine rate). 3. Obtain approximately 100 ml of 3% hydrogen peroxide in a beaker. 4. Fill 5 vials, on microtiter plate, with 3% hydrogen peroxide (substrate). Set the plate aside. 5. Obtain 5 plastic cups and label with the enzyme dilutions listed in table 1. Table 1 Enzyme Concentrations Enzyme Dilutions Enzyme Cold dH 2 0 100 units/ml 20 ml 0 ml 75 units/ml 15 ml 5 ml 50 units/ml 10 ml 10 ml 25 units/ml 5 ml 15 ml 0 units/ml 0 ml 20 ml 6. Set up a cold water bath and obtain about 50 ml of enzyme stock solution in a plastic vial. KEEP YOUR STOCK SOLUTION IN THE COLD WATER BATH AT ALL TIMES!