Reference no: EM133418348
Case: Almost all reactions in the body are controlled by enzymes. Many different enzymes are required to break down food (carbohydrates, fats, and proteins) into forms usable by the cells. Other enzymes are necessary for the cells to synthesize their own carbohydrates, fats, and proteins. Enzymes are special types of protein whose function is to control cellular reactions. These reactions occur very quickly. Speed is necessary to maintain constant cellular conditions. Enzymes are used over and over again by the cells to catalyze life-sustaining reaction. Enzymes are very sensitive to their local surroundings. Changes in pH, temperature, ion concentration, and types of ions or molecules in solution drastically alter the enzyme's ability to catalyze a reaction. Enzymes are also very specific in catalyzing only one reaction out of the millions that are possible. An enzyme is a protein and is built of amino acids. Each enzyme has a specific three dimensional structure. This structure contains a pocket called,the active site where the reaction occurs. The enzyme and substrate (reactant) combine to form the enzyme substrate complex. The substrate, activated by the enzyme in the complex, reacts to form the products, regenerating the enzyme. Various forces (hydrogen-bonding, salt bridges, disulfide linkages, etc.) hold the enzyme in its specific shape. These forces can be overcome by denaturants, which inactivate the enzyme. One such denaturant is heat. A heated enzyme (boiled) loses its three-dimensional shape and becomes inactive or denatured. Another method of inactivating an enzyme is to introduce a chemical inhibitor capable of specifically binding to the active site of the enzyme. These inhibitors prevent normal substrate-enzyme interactions. In this experiment we will investigate an enzyme called catalase. Catalase catalyzes the reaction: 2H2O2 2H2O + O2 (gas) Hydrogen peroxide, H2O2, is found in many cellular locations and is toxic above a certain concentration. Therefore, the removal of hydrogen peroxide is important to the cell's survival. Catalase is found in high concentrations in red blood cells and in many plants. The catalase for this investigation is extracted from bovine liver tissue. We will study the effect of variations of temperature on catalase activity. In order to determine the enzyme's activity (assay procedure), we must measure either the loss of a reactant or the increase of a product. In this experiment, we will measure the amount of oxygen produced during the reaction.
Description of the experiment
The catalase* for this experiment is extracted from bovine liver tissue. We used a 0.05% catalase solution. The extract should be kept on ice to keep it from decomposing until you are ready to use it. The volume of oxygen produced from hydrogen peroxide can be determined by measuring the amount of water displaced. The oxygen produced from the hydrogen peroxide decomposition is bubbled into the gas collecting tube and displaces water. The volume of gas collected is measured by the graduations on the tube.
Using a clean 10ml pipet, we added 10.0 ml of 3% hydrogen peroxide into a test tube. We put the test tube in a 250ml beaker filled with ice water and let it set for at least 3 minutes to allow the hydrogen peroxide to reach temperature of ice water. We recorded the EXACT temperature of the water bath. Using a second clean 2ml pipet, we added 1.0 ml of the catalase solution into the cooled hydrogen peroxide and mix quickly. With the test tube in the ice-water, we inserted the collection tube stopper into the test tube and immediately start timing. We shacked the test tube occasionally. Then we recorded the volume of oxygen collected after our standard time interval as determined. We also recorded the volume on data sheet. We repeated the above procedure with a clean test tube and using the following water bath temperatures: (10°C-15°C), (20°C-25°C), (30°C-35°C), (40°C-45°C).
Questions and Problems
1. Describe the effect of temperature on the activity of the catalase?
2. According to your data, at what temperature is bovine catalase at its optimal activity? Explain
3. Did the boiled catalase behave differently than the non-boiled catalase? Explain.
4. What conditions needed to remain constant for each trial?