In this series of laboratory experiments, my lab partner and I were to conduct an experiment about the oxidation rate of the enzyme peroxidase in the presence of its substrate guiacol. Also we used other substrates, such as hydroxylamine an enzyme inhibitor, to observe the weather the reaction rate was slowed down, sped up, or stopped reactions all together. These results were recorded by taking the materials in a test tube, then inserting them into a spectrophotometer to record the oxidation (intensity of color change from clear to reddish-brown) over a course of two minutes to observe color change. After all of the experiments that we ran we could conclude the enzyme oxidation rate (mmoles/min) depending on the amount of each product that was used in a single cubit test tube.
The following materials and Concepts were used to achieve oxidized/non-oxidized guaiacol:
* Boiled peroxidase (show the results of denatured enzymes/proteins when guaiacol is added) * Guaiacol (toxic substrate, common byproduct of cellular respiration) * Hydroxylamine (an extremely toxic carcinogen that is an enzyme inhibitor to peroxidase) * Peroxidase (enzyme from Turnip Root)
* PH buffer of PH₅
* Spectrophotometer (record results of oxidation rate over two minutes.
The main objective of this lab was to observe the activity of enzyme peroxidase in real time under different experimental conditions. To see how peroxidase reacts with its substrate guaiacol under different conditions. We measure the amount of substances per test tube and then combine all of them together. Each test tube contains a measured amount of any listed substances including H₂O. Each measured amount of peroxidase along with its substrate guaiacol, and other listed substances will show how actively the enzyme oxidizes the substrate. To measure the amount of oxidized substrate (amount of Hydrogen and electrons removed from guaiacol). As the measured substances are quickly and properly added to the test tubes, the test tube is then quickly inserted into the spectrophotometer. Then the results of oxidized guaiacol was measured and recorded over a course of two minutes. This allows us to digitally use precise measurements on how intense the color oxidation has become. Peroxidase, a common enzyme within many forms of life, helps catalyze the detoxification reaction of H₂O₂. H₂O₂ is a common toxic byproduct of cellular respiration. One such substrate of H₂O₂ is guiacol, a compound that my partner and I used during this experiment. As a result of the guiacol being oxidized, a reddish-brown color is formed indicating oxidized guaiacol has been formed. However, if a substrate inhibitor such as hydroxylamine inhibits the guaiacol by competing for the activation site most likely preventing color change. Therefore, depending on the amount of peroxidase, hydroxylamine, and guaiacol in a cuvette, it will determine activity of guaiacol oxidation. Each experiment required a specific amount of substances within a cuvette. After each test tube is filled with all of the samples, the cuvette is immediately covered with perafilm and inserted in the spectrophotometer to be recorded for guaiacol oxidation rate over two minutes (A/min). Once the (A/min) was recorded, those results were then calculated to find the graph equation of reactions per minute. By finding the equation we took the change in 20 seconds multiplied by 3(seconds) in order to give us our results on graphs. Experiment one was to measure the oxidation depending on the concentration of peroxidase in five separate cuvettes. Also, it was to see what the effects of a concentrated amount of denatured enzymes (boiled enzyme) has on its substrate in a separate cuvette. The enzyme solutions were prepared as described on page 5 of the Lab Handout. Experiment two was to...