Biology lab
Introduction:
In this lab, “Determining the Properties of an Enzyme”, the objectives were to observe the activity of an enzyme in a tissue extract using a spectrophotometer, and to test whether the amount of the enzyme influences the rate of the reaction, whether the temperature of the solution influences the activity of an enzyme, or whether the pH of the solution influences the activity of the enzyme. Thousands of chemical reactions occur in a cell each minute, these reactions are not random events but are controlled by biological catalysts called enzymes. Like all catalysts, enzymes lower the activation energy, or the amount of energy needed to trigger the reaction. Most enzymes are proteins with individual shapes determined by their unique amino acid sequences. The shape of an enzyme especially in its active site, determines its catalytic effects. The active site of each type of enzyme will bind only with certain types of molecules. A molecule that binds with an enzyme and undergoes chemical modification is called the substrate of that enzyme. Often metallic ions aid in the binding process, vitamins or other small molecules that are called co-factors or coenzymes. The binding between enzyme and substrate consists of weak, non-covalent chemical bonds, forming and enzyme-substrate complex which only exists for a few milliseconds. During this instant, the covalent bonds of the substrate either come under stress or are oriented in such a manner that they can be attacked by other molecules. The result is a chemical change in the substrate that converts it to a new type of molecule called the product of the reaction. The product leaves the enzyme’s active site and is used the by the cell. Individual enzyme molecules may enter the catalytic cycle several thousand times per second; thus, a small amount of enzyme can convert large quantities of substrate to product. Eventually enzymes wear out, break apart and lose their catalytic capacity. Cellular proteinases degrade inactive enzymes to amino acids, which are recycled by the cell to make other structural and functional proteins. When no enzyme is present, the chemical reaction catalyzed by the enzyme does not occur at an appreciable rate. Conversely, if enzyme concentration increases, the rate of the catalytic reaction associated with that enzyme will also increase. The pH or salt concentrations of a solution affect the shape of enzymes by altering the distribution of + and – changes in the enzyme molecules which, in turn, alters their substrate-binding efficiency. Temperature, within the physiological limits of 0˚ and 40˚ , affects the frequency with which the enzyme and its substrates collide and, hence, also affects binding. All factors that influence binding affect the rate of enzyme-catalyzed reactions. In this lab the enzyme peroxidase was used. Peroxidase is a large protein containing several hundred amino acids and has an iron ion located at its active site. The source of this protein, peroxidase, in this lab was horseradish root, though horseradish root contains hundreds of different types of enzymes, including peroxidase, only peroxidase will react with H2O2. The normal function of peroxidase is to convert toxic hydrogen peroxide (H2O2) into harmless water (H2O) and oxygen (O2). The peroxidase reaction can be measured by following the formation of oxygen. The amount of oxygen present after the reaction can be measured either by the accumulation of gas in a closed system connected to a manometer or by the appearance of chemically active oxygen. Many dyes will react with active oxygen by changing from a colorless to a colored state, such tests are called dye-coupled reactions, and were used in this experiment. The dye used was guaiacol, which turns brown when oxidized. To measure the amount of brown color in the final product, the enzyme, substrate, and dye can be mixed in a tube and immediately placed in a spectrophotometer. As color accumulates, the