Methods
To properly perform our experiment, we needed to have catechol (the substrate), catecholase (the enzyme), and water to allow the reaction to occur in. We also needed some means of manipulating the temperature, and some way to determine the rate of the reaction. Our method of finding the reaction rate was to time the experiment, and test the amount of product formed in that time using a spectrophotometer. The spectrophotometer can tell us the amount of product formed because the product has a pigment, and the spectrophotometer sends a beam of light into the tube, and measures how much of it is absorbed by the pigment in the product. But in order to get accurate results, we had to find a way to have the spectrophotometer not count the pigment in the enzyme when it measured the light absorbed. To do this we had to make a “blank” tube. The blank tube contained the water and the catecholase, but did not contain the catechol. This allowed us to measure the pigment in the catecholase without allowing the reaction to start. By using this tube to zero out the machine before each measurement, we were able to get accurate readings of how much product was formed. Another way worked towards greater accuracy was to run four trials of each temperature tested. This allowed us to compare the results from each trial within a temperature and watch out for any abnormal readings. So, we set up the blank tube to contain 5.5 mL of water and .5 mL of enzyme, and all of the trials were set up as two tubes to begin with. One tube had 3.5 mL of water and 2.0 mL of catechol, and the other had .5 mL of enzyme. By keeping the enzyme and substrate separate, we were able to have more control over the starting point of the reaction, and were able to bring each individual component to the desired temperature before starting. The temperatures that our group tested were 37oC, 45oC, 50oC, 55oC, 65oC, and 75oC. Both our group and group A-4 tested 37 and 45 degrees. This allowed us to compare each group’s results more accurately.
The setup for the actual experiment was that each temperature had its own test tube rack. Four tubes of water and catechol and four tubes of enzyme were put into each rack, and these racks were then submerged into either a hot water bath or a circulating water bath, depending on which temperature was being tested. The tubes were allowed to sit in the water for approximately five minutes to allow the contents of the tubes to come up to the temperature we were trying to test. Then the tube of enzyme was poured into the tube of catechol, the tube was covered and inverted to mix the contents, and the timer was started. We allowed the reaction to run from three to five minutes, keeping track of exactly how long each set went for. Our method for ensuring that the reactions ran for the exact length of time stated was to start each reaction thirty seconds later than the first, and then take the measurements from the spectrophotometer exactly thirty seconds apart, keeping the tubes in the same order. Before measuring each tube, we again inverted it several times to make sure that the product was uniformly distributed throughout the tube.
Results
Results for Group A-5
(Table 1)
Results for Group A-4
(Table 2)
(Graph 1
(Graph 2)
According to our results, the optimal temperature for the reaction of catecholase is 45oC. We were able to determine this because the average reaction rate was highest at this temperature (see table 1). We were correct in assuming that the reaction would gradually speed up as the temperature increased until it reached a point where the enzyme began to denature, and then the reaction rate would slow after that. This gradual increase, peak, and then decrease can be seen in the graph 1 above. The bars above and below each data point are representations of the standard deviation associated with each value. This shows how much variation we had within each set of trials. When we compared our results to group A-4, it was clear that they too found the peak reaction rate to be at the 45-degree temperature. This served to further verify our results. It is interesting to note that in the graph of both of our data, although the actual values are not the same, the overall conclusions are.
Discussion
After analysis of the data, it is clear that our hypothesis was correct, because the found peak does fall within the range of 23 and 75. We can be fairly certain that our data are correct because we were able to control the fixed variables, such as the amounts of each component of the reaction, very well by having the same person measure out the same thing for each trial. We were fairly accurate at controlling the temperature because the baths were monitored at a steady temperature for the time the reaction was taking place, although it would have probably been to our benefit to actually take the temperature of the liquids inside the test tubes instead of only monitoring the environment outside of the tubes. Also, we could have increased accuracy by having the spectrophotometer nearer to the baths, because there was some heat lost during transport, which could have affected the tubes that were measured later. There was also some confusion at the beginning of the experiment about exactly what technique was to be used, which resulted in our having to repeat the trials for the 65-degree temperature. We assume that similar problems are to blame for the differences in the reaction rates for 37o and 45o between our group and group A-4. We also discovered after the experiment had been completed that the other group had left the catechol out in a separate tube during heating, while we left the enzyme out. This difference in method may have contributed to the differences.
To make sure that these problems did not result in faulty conclusions, I looked up another experiment on the effects of temperature done by Pierre Greenway. Greenway’s findings indicate that the peak temperature for enzyme function is actually at 40oC, and not 45. This is an interesting discovery, since we did not test thoroughly in the range of 37o-45o. The next course of experimentation suggested by these findings would be to thoroughly test the reaction rates at temperatures between 35 and 45 degrees Celsius. Measuring at one-degree intervals would give the best results, but may require another collaboration to be achieved in a reasonable amount of time. It may also be interesting to test the reaction rate at even higher temperatures, and try to find the point at which all of the enzyme is completely destroyed. According to the results of Greenway, this temperature was around 60oC for him, but we tested beyond that in our own experiment and found the rate to still be decreasing. Any of these questions would be interesting to try to answer.
