Enzymes Lab Report

Title: The Effect of Varying Amounts of Substrate and Enzyme on a Reaction Rate

Abstract In living organisms, certain reactions must take place rapidly to assist life. This occurs because of enzymes, because all reactions would take place too slowly to sustain life (Jacklet, 237). Enzymes are large protein molecules that catalyze specific chemical reactions without being used up in the process. Each enzyme has a region on its surface, called the active site, which recognizes a specific substrate molecule. The substrate is chemically attached to the active site and binds an enzyme-substrate complex. With the use of a spectrophotometer, the absorbance is recorded and the rate of reaction is observed when differing amounts of substrate and
enzyme are added.
Introduction
Whether we can see it with the human eye or not, each living cell in an organism must carry out complex reactions to stay alive.
These activities are only possible because of the presence and activity of enzymes. Each enzyme reacts with a substrate molecule, and during the reaction, the substrate is broken down to products which are then released. Varying the enzyme concentration, as well as varying the amount of substrate, can change the rate of the reaction. A greater number of enzyme molecules mean more active sites, which will speed up the reaction as they bind with substrate molecules. As the substrate increases, the same can be said, up to a certain concentration known as the saturation point. According to these enzyme processes, it can be assumed that as the amount of the enzyme increases as the substrate remains constant, the absorbance will increase. Likewise, it can be assumed that as the amount of substrate increases as the enzyme stays constant, the absorbance will increase as
well.
Materials & Methods In the lab experiment, scientists prepared different solutions for enzyme testing. The materials used were a spectrophotometer, pipettes of different volume, test tubes, and the various substances used to make the solutions. A spectrophotometer measures what fraction of the light passes through the solution and indicates the amount of light absorbed compared to that absorbed by a clear solution. The enzyme used in the experiment was peroxidase, which catalyzes the breakdown of hydrogen peroxide to water and oxygen, which each cell uses for metabolism. The peroxidase was obtained by a turnip extract mixed with water. The components of the solution are all clear, so it’s impossible to see the reaction without this indicator, which is added to the solution. In this case, guaiacol is used, which changes color when it reacts with the oxygen in the solution, allowing the reaction to be seen. In experiment one, which is the baseline test, after the control is made in the first tube, two tubes are made for testing of absorbance. In the second tube, 0.1 ml of guaiacol, 0.2 ml H2O2, and 4.7 ml dH2O were added. In the third tube, 1.0 ml turnip extract and 4.0 ml dH2O were mixed together. Tubes two and three were mixed together, and within twenty seconds of mixing, put into the spectrophotometer. The absorbance was observed every twenty seconds for the next two minutes, and then graphed as the baseline experiment which all future experiments would be based. In experiment two, the same amounts of each substance are added, except the amount of enzyme is changed. For the first test, twice the amount of the enzyme was used, and for the second, half of the amount of enzyme was used, keeping every other component constant. In experiment three, five new tests are made. Compared to the baseline found in experiment one, varying amounts of substrate is added this time. In the second test after the baseline, twice the amount of substrate is added. In the third test, half the amount of substrate is added. This continues for three more tests, adding ¼ the amount of substrate, four times the amount of substrate, and 1/10 the amount of substrate, with all other variables remaining constant. The same steps with the spectrophotometer are used in each test, mixing together the two vials and measuring the change in absorbance when the substrate or enzyme is changed within twenty seconds of the reaction. The results are then graphed, and the slopes for each line from experiment two are plotted on another graph. From these points, the maximum rate (Vmax) is found after plotting the points. Once the maximum rate is found, the Km, or Michaelis-Menten constant, is found by taking half of the Vmax value.

Results
(See attached data and graphs for in-depth results of findings)
Graph 1: For the results of experiment one, the graph depicts the absorbance of our baseline test, two times the enzyme, and half of the enzyme.
Graph 2: For the results of experiment two, the graph depicts the absorbance of the baseline test, two times the substrate, half the substrate, a quarter of the substrate, four times the substrate, and a tenth of the substrate.
Graph 3: The third graph depicts the rate of reaction vs. the concentration of the substrate. From this graph, the Vmax and Km are determined. In our results, the Vmax was equal to .00325, and the Km value was the closest to 1/10 times concentration of the substrate.
Discussion
The results of the experiments change according to what was expected. When the enzyme concentration is changed, the reaction rates change accordingly. In the first experiment, when the enzyme amount is doubled, and reaction rate is doubled as well in comparison to the baseline test. When the enzyme concentration is halved, the reaction rate is halved in comparison to the baseline test. When the substrate concentration is changed, the results change along the same trend. As the substrate concentration gradually increases, the reaction rate increases, and as the substrate concentration gradually decreases, the reaction rate decreases. In our personal results, the baseline test was not consistent into the second experiment. This may be due to human error during the baseline test, or a mistake with the equipment used. Still, in both cases, the graph is an increasing curve, showing the gradual increase in the reaction rate when each substrate or enzyme is changed. The results shown prove the hypotheses stated above. Enzymes increase the reaction rate in chemical reactions, so when the enzyme content is increased in any way, the reaction rate increases more rapidly, and when it is decreased, the reaction rate increases slower in comparison to the baseline. When the substrate is increased, because of the increasing number of molecules the enzymes can attach to, the reaction will increase faster, and when the substrate concentration is decreased, the reaction rate will increase faster until it hits its maximum rate, and when the substrate concentration is decreased, the reaction rate will increase less rapidly. Although the reaction rate slowed down and increased less fast as the amounts got smaller, the reaction rate never decreased on any of the tests, because of the positive slope on each of the plots. According to our results, as stated above, the baseline does not hold up in the comparisons on the second experiment. The baseline test has a smaller slope than ½ times the substrate, which shouldn’t be the case. As stated above, this is due to human error or faulty equipment, and scientists can further study the validity of these claims by doing the experiment a second time to prove the hypotheses true. Scientists could also test other substances that could possibly cause the reaction rate to decrease, and analyze the properties that caused the decline in the reaction rate. Through these tests, further discoveries could be made about enzymes and reactions and how they benefit everyday chemical processes, whether we can see them with the naked eye or not.

Literature Cited
Investigations in Modern Biology. 7th ed. Colorado: Morton Company, 1996. 237-242.