For this lab exercise, each group was assigned the task of choosing some aspect of enzyme function and studying it by designing an original experiment. Before choosing a specific aspect to study, all the lab groups did a general experiment to study the reaction rates of a specific enzyme, catecholase. Catecholase is an enzyme found in some many fruits and vegetables, and is responsible for the browning that occurs when a fruit is cut or damaged. Its substrate, catechol, reacts with oxygen in the air to produce water and benzoquinone. This is what has the reddish brown color.
This experiment became the impetus for our decision as to what we wanted to study. When we compared our results to the results of the other groups in our lab section, we discovered that our results were unusually high. After discussing this for some time, we determined that the most probable reason for this is that one of the group members had held the test tube with the reaction occurring inside in her hands while waiting to use the spectrophotometer. The implementations of this were that we knew that this specific enzyme could work more efficiently at a higher temperature.
This makes sense because we know that when the temperature increases, there is more movement at the molecular level, and molecules of enzyme and substrate become more likely to collide, making the reaction occur more often. However, we also understood that after a certain point, a high level of heat would change the shape of the enzymes, thus denaturing them, or making them nonfunctional. This raised an interesting question? For this enzyme, what is the optimal temperature for function-that is, at what temperature are the molecules colliding as much as is possible without allowing the heat to change the shape of the enzyme?
We knew that this temperature had to be above room temperature, and we conjectured that it would be below 75oC, because we couldn’t realistically test our hypothesis much farther beyond that point in the limitations of time and safety. To help with the limits of time, we combined to work with another lab group, group A-4, to cover all of the temperatures required. 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 .