Investigating Enzyme Activity: Amylase and Catalase Experiments

Factors Affecting Enzyme Activity Introduction Enzymes are globular proteins that act as catalysts for biological reactions. An enzyme will speed up a reaction rate without being consumed. All biochemical reactions are catalysed by enzymes. An enzyme works by binding to a specific reactant (called the substrate) via a complementary indentation called the active site. According to the induced fit model, the active site undergoes a conformation change to better adhere to the substrate and this stresses the substrate’s bonds, catalysing its conversion into a product. Enzymes are also very specific – they only act on one substrate (or a class of related molecules). Typically, only one substrate will fit into the active site. EXPERIMENT 1: Amylase and Starch Amylase is an enzyme found in both saliva and the digestive juices of the intestines. It catalyses the hydrolysis of starch (a polysaccharide) into shorter subunits (dextrins or maltose). The extent of the hydrolysis depends on how long the amylase is allowed to react. If the starch is hydrolysed completely, the resulting product is glucose (a monosaccharide). Iodine forms a blue-black complex when reacted with starch but does not react with glucose. If iodine is added to a glucose solution, the only colour seen is the brown-yellow colour of the iodine. Hence, the activity of amylase can be measured according to the colour of a starch solution once iodine is added. If the solution turns blue-black, the amylase activity is low (starch has not been hydrolysed); whereas if the solution turns brownish-yellow, the amylase activity is high (all the starch has been hydrolysed). Methodology 1. 2. 3. 4. 5. 6. 7. 8. Set up three test tubes in a test tube rack (label 1 – 3). In tube 1, add 4ml of 1% starch solution and 4ml of amylase solution. In tube 2, add 4ml of 1% starch solution and 4ml of deionised water. In tube 3, add 4 ml of deionised water and 4 ml of amylase solution. Place the test tube rack in a 37ºC water bath for 10 minutes. Remove tubes from water bath, add three drops of iodine solution to each tube and mix. Transfer contents to a cuvette and measure the absorbance of green light with a colourimeter. Record your observations. EXPERIMENT 2: Catalase and Hydrogen Peroxide Hydrogen peroxide is produced as a waste product in every cell of the human body. It is a byproduct of many normal chemical reactions. If the cells did not break down the hydrogen peroxide, they would die. In cells, the enzyme catalase works on its substrate hydrogen peroxide converting it to water and oxygen. When paper discs are soaked in catalase and then immersed in a solution of hydrogen peroxide, the catalase-soaked disc begins to produce oxygen gas which causes the disc to float. The time taken for the disc to resurface can be used to indicate the rate of activity (high activity = less time). Methodology 1. 2. 3. 4. 5. To a 200ml beaker, add 100ml of 1% hydrogen peroxide solution (beaker 1). To a second 200ml beaker, add 100ml of 0.5% hydrogen peroxide solution (beaker 2). Using a hole puncher, punch 6 paper discs from filter paper. Using a pipette, add 1ml of 0.05% catalase solution to a well of a multi-well plate. Using forceps, submerge a paper disc in the catalase solution for 10 seconds, then drop the disc on the surface of the hydrogen peroxide in beaker 1. 6. Begin timing as soon as the disc touches the surface and measure how long it takes (in seconds) for the disc to resurface. 7. Repeat steps 5 and 6 for beaker 2. 8. Conduct three trials in total for each beaker and record your observations.