Enzyme Activity - Lab Report Example

Enzyme Activity Introduction Enzymes are biological catalysts that are used in hastening biochemical reactions. Without the presence of enzymes, most chemical reactions in the body such as respiration would take a very long time to come to proceed. In addition, a lot of heat would be generated and lead to the destruction of living cells. Enzymes increase the rates of these reactions by lowering the activation energy of the reactions. Activation energy can be described as the least amount of energy that is needed for reacting substances to go through a reaction. An enzymatic reaction involves three main components namely the enzyme, the substrate (the substances that undergo the reaction), and the product. Enzymes are protein molecules with a three-dimensional shape that includes an active site whose shape is complementary to the shape of the substrate. For this reason, each enzyme is specific to the type of substrate and reaction that it can catalyze. Several factors affect the rate of enzyme activity. They include temperature, enzyme concentration, substrate concentration, and pH. Enzymes, being made of proteins, are sensitive to factors that affect the structure and function of proteins. Therefore, each type of enzyme has an optimum range of temperature and pH at which it exhibits maximum activity. Any deviation from the optimum pH of an enzyme leads to interactions of the charged groups of the amino acids in the active site of the enzyme, which leads to a distortion of the enzyme active site that consequently lowers catalysis. For example, the enzyme catalase is responsible for the breakdown of hydrogen peroxide into oxygen and water. Therefore, the amount of oxygen produced is proportional to the activity of catalase. Hence, it is important to measure the amount of oxygen produced to measure the activity of catalase. The purpose of this lab is to determine the influence of pH and temperature on the activity of the enzyme catalase. Hypotheses 1. HO: Various temperatures will not affect the rate and amount of oxygen produced when hydrogen peroxide is broken down by catalase. HA: Various temperatures will affect the rate and amount of oxygen produced when hydrogen peroxide is broken down by catalase. 2. HO: Various pH conditions will not affect the rate and amount of oxygen produced when hydrogen peroxide is broken down by catalase. HA: Various pH conditions will affect the rate and amount of oxygen produced when hydrogen peroxide is broken down by catalase. It is expected that different environments of pH and temperature will affect the activity of catalase because they will influence the structure of the active site of the enzyme hence alter catalysis. Methods and Materials Three test tubes were filled with 4ml of catalase and 2ml of distilled water while three other test tubes were filled with 5ml of hydrogen peroxide. The tubes containing peroxide and catalase were labeled as 2oC, 37oC and 80oC. A plastic tub was then filled with cold tap water to an approximate depth of 3 to 4 inches after which the temperature of the water was adjusted to between 37 and 40 oC using warm water. 50ml graduated cylinders were then prepared by submerging them in the water tubs and allowing them to fill without any air bubbles. The cylinder was then put upside down with its mouth submerged. The cylinder was then put in a ring stand and secured in position with the mouth just below the surface of the water. A reaction chamber was then prepared by soaking three paper disks in catalase incubated at 37 oC and placing them on one wall of the chamber. 5ml of the 37 oC incubated hydrogen peroxide was then added to the chamber ensuring that the peroxide did not make contact with the discs. The neck of the chamber was closed with a stopper after which the chamber was held in the tub for approximately 2 minutes to equilibrate to the tub water temperature. The rubber tubing was then pinched closed, and the chamber was submerged on its side with the paper discs in the top position. The rubber tubing was then inserted into the inverted 50ml cylinder without disturbing the water column. The reaction chamber was then rotated to create contact between the discs and the peroxide after which the reaction was initiated by shaking the chamber. The oxygen trapped in the 50ml cylinder was then recorded every 30 second. A similar procedure was repeated for the 2oC and 80oC by setting the water baths at 2oC and 80oC using ice and hot water respectively. The volumes of oxygen produced were recorded at intervals of 30 seconds in Table 1. To assess the effect of pH on catalase activity, the above setup was repeated at a temperature of 37oC. However, the paper discs were soaked in catalase solutions exposed to pH conditions of 2, 7 and 10. The amount of oxygen produced was also recorded at intervals of 30 seconds and recorded in Table 2. Results Table 1: Temperature Effects on the Hydrogen Peroxide/Catalase Reaction (ml of Oxygen produced over time) Time (sec) Temperature (oC) 2 37 80 0 0 0 0 30 7 6 0 60 12 12 0 90 17 15 0 120 21 22 0 150 24 24 0 180 29 27 0 210 32 28 0 240 35 30 0 270 38 31 0 300 40 33 0 330 41 34 0 360 42 35 0 390 45 36 0 420 46 37 0 450 47 38 0 480 48 38.5 0 510 49 39 0 540 49 39.5 0 570 50 40 0 600 50 41 0 Figure 1: A graph of the effect of temperature on the activity of catalase Table 2: Effects of pH on the Hydrogen Peroxide/Catalase Reaction (ml Oxygen produced over time) Time (sec) pH 2 7 10 0 0 1 0 30 7 9 2 60 13 15 6 90 17 20 8 120 19 25 11 150 21 29 13 180 23 32 16 210 24 35 18 240 25 38 20 270 25 40 21 300 26 42 24 330 26.5 44 26 360 27 45 27 390 27 46 29 420 27 47 31 450 27.5 48 31 480 28 49 34 510 28 49 35 540 28 50 36 570 28 50 37 600 28 51 48 Figure 2: Effects of pH on the hydrogen peroxide/catalase reaction Rate of reaction for various conditions 2oC: Maximum rate=0.175 ml/sec ; Average rate=0.083 ml/sec 37oC: Maximum rate=0.2 ml/sec; Average rate=0.0683 ml/sec 80oC: Maximum rate= 0ml/sec; Average rate=0ml/sec pH 2: Maximum rate=0.230 ml/sec ; Average rate=0.047 ml/sec pH 7: Maximum rate=0.3ml/sec ; Average rate=0.085 ml/sec pH 10: Maximum rate=0.367 ml/sec ; Average rate=0.080 ml/sec It was observed that the rate of reaction of catalase varied at different temperatures (Table 1). The reaction was highest at 2oC while the lowest reaction rate was observed at 80 oC as indicated in Figure 1. The activity of catalase also varies with changes in pH as recorded in table 2. The highest activity was obtained at pH 7 whereas the lowest activity was recorded at pH 2 (Figure 2). Discussion The purpose of this lab report was to investigate the effect of temperature and pH on the activity of catalase. It was hypothesized that various temperatures would influence the activity of catalase. It was also hypothesized that various pH values would affect the activity of catalase. Therefore, from the obtained results, it was confirmed that temperature and pH influenced the activity of catalase. The highest activity was observed at a temperature of 2 oC while the lowest activity was recorded at 80 oC. At 80oC, the temperatures were too high leading to the denaturation of the enzyme, which distorted the structure of the active site of catalase hence there was no activity at all as seen by an average activity of 0ml/sec. A temperature of 2oC provides the optimum conditions for the activity of catalase because the highest activity was recorded at this temperature as seen in the average activity of 0.083 ml/sec. pH influences the activity of catalase by altering the ionic configuration of the amino acids in the active site of the enzyme. At acidic and alkaline pH, the activities of catalase were low at 0.047ml/sec and 0.080ml/sec. The highest activity of catalase was observed at a pH of 7 (0.085ml/sec). Therefore, the findings of this lab show that the best conditions for the activity of catalase are temperatures of 2oC and a pH of 7. If more catalase is added to the 37oC solution without the addition of peroxide, there will be no change in the rate of the reaction because all the peroxide would already have been broken down into oxygen and water. However, if more peroxide is added then the reaction will proceed, and more oxygen would be produced because enzymes are not consumed in the reactions that they catalyze. Read More