Abstract:
Enzymes are proteins and they can be protein catalysts that speed up the rate of chemical reactions. Enzymes are important because almost all chemical reactions in living cells need to be speed up or they would occur too slowly. The speed of enzyme-catalase reactions can be affected by factors such as temperature and substrate concentration. In these experiments, two hypotheses were tested. One was that as the concentration of the substrate (H2O2 in this experiment) increases, the rate of reaction will speed up until it reaches the saturation point. The other was that as the temperature increases, the reaction rate will increase, until it becomes too extreme. To test both hypotheses, the amount of time to produce 10 mL of O2 was measured. 10mL of concentrated substrate was mixed with 10mL of catalase and recorded the time. For experiment #1, this was done 5 times with varying concentrations: 0.0%, 0.1%, 0.2%, 0.4% and 0.8%. For experiment #2, catalases of differing temperatures were used: boiled (100C), warm (37C), room (25C), and ice cold (0C). The first experiment showed that the reaction rate increased as the substrate concentration increased. For example, from 0.2% to 0.4% concentration, the average rate of reaction increased from 2.90 to 28.76 mL/min. This was likely because if there is more of a substance, more enzyme-substance collisions will occur which will lead to an increase in the reaction time. In the second experiment, an initial increase was seen, but steady decrease was seen as temperature continued to go up. For example, from cold to room temperature, the average rate of reaction increased from 35.24 to 70.97 mL/min and then decreased at warm temperature to 33.03 mL/min. This result is likely because as the temperature increases, the enzymes will move faster, which will also cause more collisions. However, once the temperature is raised past a certain point, the reaction rate will plateau due to the enzymes becoming denatured. In short, both of the tested hypotheses were supported.
Introduction:
One of the main functions of proteins is enzymatic activity. Enzymes are catalysts that speed up chemical reactions in cells. Enzymes catalyze reactions by lowering the activation energy required to begin a reaction (Cooper 2000). Catalase is an enzyme that decomposes hydrogen peroxide (Baureder et al 2014) and is found in the liver (Glorieux & Calderon 2017). Temperature, pH, and substrate concentration can affect enzymatic activity (Eed 2013). In these experiments, we wanted to know how things, such as temperature and substrate concentration, can affect the rate of reaction of an enzyme-catalase reaction. Therefore, for the first experiment, it was hypothesized that as the concentration of substrate increases, the rate of reaction will increase because the more substrate present, the more collisions and bindings that will take place. The null hypothesis to this is that an increase in the substrate concentration will have no effect on the rate of reaction. For the second experiment, it was hypothesized that as the temperature of the catalase increased, the rate of reaction will increase because an increase in temperature will make the substrate move faster, which will also result in more collisions and binding. However, it was also predicted that once the temperature increased past a certain point, the enzyme would become denature, the active sites would be destroyed, and the reaction rate will plateau. The null hypothesis to this is that a change in temperature will have no effect on the rate of enzyme-catalase reaction.
Materials and Methods:
For both experiment 1 and 2, a 600 mL beaker was filled with 400 mL of water. Then a 10mL graduated cylinder was filled to overflow with water. The opening of the graduated cylinder was sealed with one of the scientist’s fingers so when it was immersed in the 600 mL beaker, so no oxygen gas would enter the graduated cylinder. Then, the short end of a U-shaped glass tube inside the 10 mL graduated cylinder. After setting up, the experiments were started. 10 mL of substrate (H202) and 10 mL of catalase were put in a 50 mL Erlenmeyer flask and then the rubber stopper of the U-shaped tube was placed in that flask. While one partner swirled the flask with the catalase and the substrate, the other watched for the reaction and with a timer app on their phone, timed how long it took for 10 mL of oxygen to be produced. This experiment was done six times with different concentrations of substrate: A control group, 0.0%, 0.1%, 0.2%, 0.4%, and 0.8%. The beaker and graduated cylinder were rinsed every time there was a change in concentration. The set up and procedure for experiment was the same except it was performed four times with catalase of varying temperatures: 0C, 25C, 37C, and 100C. Microsoft Excel was used to create tables, create graphs, and perform a statistical analysis of the results. To determine if any of the temperature differences were significant, a t-test with a cutoff value of 0.05 was used.
Results:
Figure 1-This table shows the group by group data for the rate of reaction with various substrate concentrations
Figure 2-This table shows the group by group data for the rate of reaction with various catalase temperatures.
Figure 3-This table shows the p values for the difference in temperatures.
Figure 4-This graph shows that class averages for the rate of reaction for varying substance concentrations (0.00%, 0.10%, 0.20%, 0.40%, 0.80%). Error bars represent one standard deviation.
Figure 5-This graph shows the class averages for the rate of reaction for varying catalase temperatures (Boiled, Warm, Room, and Ice Cold). The error bars represent one standard deviation.
In experiment 1, the average rate of reaction for the substrate concentration of 0.0% was 0 mL/min, for 0.1% it was 1.3125 mL/min +-.78, for 0.2% it was 2.90 mL/min +- .52, for 0.4% it was 28.76 mL/min +-14.1, and for 0.8% it was 72.2575 mL/min +-54.04 (Figure 1). In experiment two, the average rate of reaction for 0C was 35.24 mL/min +- 18.58, for 25C it was 70.97 mL/min +-54.02, for 37C it was 33.03 mL/min +-20.05, and for 100C it was .13 mL/min +-.35 (Figure 2). The p-value of room temperature vs warm temperature was .0860, for room temperature vs cold it was .09866, and for room temperature vs boiled it was .00233 (Figure 3).
Discussion:
The purpose of the experiments were to test two hypotheses: as the concentration of the substrate increases, the reaction rate will increase until it reaches the saturation point and as the temperature increases, the rate of reaction will increase until it reaches a point where the enzyme becomes denatured. In experiment 1, the data supports the hypothesis because the average rate of reaction increased as the concentration of substrate increased (Figure 4). However, for .4% and .8% concentrations, the standard deviations are large, coming out to 14.05 and 54.04 respectively. For experiment 2, the differences between room temperature and warm temperature and room temperature and cold were not significant. Their p-values were 0.086 and 0.099 respectively, and they were not less than the cut-off value of 0.05, meaning they were not significant. The only significant difference was between the room temperature and boiled catalase (Figure 3). The p-value for this difference was .002, which is less than the cut-off value of 0.05, meaning it is significant. The average rate of reaction increased from cold to room temperature but then decreased from room to warm and again from warm to boiled (Figure 5). This data supports the hypothesis. The results in experiment one occurred because increasing the substrate concentration causes more enzyme-substrate collisions which leads to binding and in turn, causes an increase in the rate of reaction. The activation energy needed for the reaction was lowered due to the enzymes and the reaction sped up (Cooper 2000). The results from experiment two likely occurred because an increase in temperature causes the enzymes and substrate to move faster, which also results in increased colliding and binding. However, a sudden drop off was seen from the room temperature catalase to the warm catalase. This is likely because somewhere in between 25C and 37C, the temperature reached a point where it denatured the enzyme, which caused the activation sites on the enzymes to be destroyed, decreasing the rate of reaction. In short, both experiments showed that enzyme catalase reaction are in fact effected by variables such as temperature and substrate concentration (Eed 2013). Possible improvements to this experiment include more accurate timekeeping and more accurate measurements. The concept could be taken further by seeing at what concentration the reaction would reach the saturation point and at exactly what temperature the rate of reaction began decreasing.
References:
Baureder M, Barane E, Henderstedt L. 2014. In vitro Assembly of Catalase. Journal of Biological Chemistry 289: 28411-28420.
Glorieux C & Calderon PB. 2017. Catalase, a remarkable enzyme: targeting the oldest antioxidant enzyme to find new cancer treatment approach. Biological Chemistry 398(10):1095-1108.
Eed J. 2013. Factors affecting Enzymatic Activity. Essai 10(19).
Cooper GM. 2000. The Central Role of Enzymes as Biological Catalysts. The Cell: A Molecular Approach 2.