The reactions and Results of Using Enzyme Turnip Root Peroxidase Lab results from: Andrew Compton, Mickey. Results published 9/29/2012. TA: 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 PH? * 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 H? O. 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.
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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 H? O?. H? O? is a common toxic byproduct of cellular respiration. One such substrate of H? O? 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 measure the effect of pH on peroxidase activity.
Using buffers pH3, 5, 7, and 9 the same amount of water, enzymes, and substrate was added to each solution. The amount of each substance is given on page 6 of the lab handout. Experiment three was to measure the effect of substrate concentration instead of peroxidase concentration from experiment one. Four test tubes are to each have different concentrations of guaiacol to see how much oxidation occurs. The amount of each substrate is given in page 7 of the lab manual. The fourth experiment required the results of oxidation levels with the presence of hydroxylamine. It was to dd specific amounts of inhibitor hydroxylamine to two cuvettes in given amounts. Ex1 test tube| 1| 2| 3| 4| 5| A/min| 0. 816| 0. 039| 1. 65| 0. 594| 0. 606| ?L Enzyme| 150| 0| 350| 50| 25| Experiment one shows that, as the enzyme concentration is increased, in A/Min. Meaning the more the enzyme the more oxidation that tends to occur. Ex 2 test tube| 6| 1| 7| 8| pH| 3| 5| 7| 9| A/min| 0. 093| 0. 816| 0. 672| 0. 021| Experiment two shows what rate of activity is shown with different levels of pH. The graph concludes that the neutral pH of 7 shows the optimal rate of A/min oxidation. Ex 3 Test tube| 1| 9| 10| 11| 12|
A/min| 0. 816| 0. 729| 0. 516| 0. 315| 1. 551| substrate ? L| 500| 300| 200| 100| 1000| In experiment three, guaiacol concentration is changed to show the different amounts of oxidation reactions or A/min. The graph concludes that cuvette twelve that contains the highest concentration of substrate. Will create the highest amount of oxidation reactions. Ex4 Test tube| 13| 14| 1*| A/min| 1. 473| 1. 758| 0. 816| Inhibitor| 500| 100| 0| Experiment number four was supposed to show the negative effect of an inhibitor on an enzyme. The experiment shows invalid results. However, if the results were accurate, the 500?
L concentration of hydroxylamine should have produced the least amount of a/min. The main objective within the experiments is to determine the activity rate of oxidation. With the instructions given, we are to predict how each reaction will occur. Weather there be a small amount of reactions, major amounts of reactions or none at all. In experiment one our results showed that the higher concentration of an enzyme the higher the activity of reactions occurred with the substrate. The experiment was to show if the concentration of enzyme would have a major effect in reaction activity if the amount of substrate stayed the same.
From the results, you can clearly see that the enzyme activity was at its most optimal when at 200uL. However, the denatured enzymes had hardly any activity because they aren’t natured proteins. Also, cuvette 3 showed that even with an extremely high concentration of enzyme it still does not have as high of activity rate because of the chances it will combine with its substrate is low because of the amount of H2O. Experiment two was to see what level of pH buffer was the most optimal for enzyme activity with its substrate. The results of the experiment showed that the enzyme was most optimal at pH 7.
Since pH 7 is a neutral pH, it shows that the other pH’s that are basic or acidic tend to hinder the activity of the enzyme activity. Weather donating H+ or adding H+. Experiment three was the opposite of experiment one. It was to see what the effects of substrate concentration had on peroxidase activity. From the spectrophotometer results, the most optimal activity rate occurred with 1000uL of substrate. With cuvette twelve being the highest amount of activity, this shows that the more substrate you have creates an even faster rate of activity with the same amount of enzyme in any cuvette.
The more substrate the more of a chance there is to react with the enzyme. Experiment four was the only one containing peroxidase’s competitive inhibitor hydroxylamine. Hydroxylamine is an inhibitor therefore it is supposed to either stop or slow the activity rate of enzymes. Cuvette 13 had a higher concentration of hydroxylamine. Therefore, the higher concentration of hydroxylamine the less enzyme oxidation activity is occurring, and more competitive inhibitors are attaching to peroxidase. Overall, these experiments show what amounts of concentrations have on enzyme oxidation activity upon its substrate, what temperature/pH is ctivity most optimal in, and what shows how much an inhibitor can affect an enzymes reactivity rate. With increasing enzyme concentration, there was more of a chance for it to come into contact with its substrate therefore increasing activity. However, enzyme activity will slowly diminish once the substrate has been oxidized. On the other hand, if you have an extreme amount of substrate. The experiment shows that the reaction rate is much higher due to the amount of oxidation occurring. These experiments had to be done a few times to receive valid results.
The spectrophotometer had varying A/min and the test called for constant changes in variation of substance measurements to receive valid results. Also, the whole laboratory experiment for experiment number four was invalid. The hydroxylamine was tainted and therefore unable to inhibit peroxidase. Therefore, giving invalid results. In this lab, we investigated how much substrate and enzyme concentration affected the rate of oxidation reaction. We investigated what the optimal pH was for basic enzyme activity/guaiacol oxidation, and what happens when an inhibitor is added to an even solution of enzyme and substrate.
We can conclude that substrate and enzyme concentrations are most optimal when one is much higher than the other. However, higher substrate concentration showed that the activity is higher due to the likelihood of reactions with its enzyme. Also, even amounts of enzymes and substrate shows highest activity rates when at a neutral pH instead of in a more basic or acidic solution. The enzyme inhibitors clearly show the slowing of activity rate when more of it is applied to a solution of enzymes and substrate.
The relationships of all of these procedures was to show us under what conditions does the oxidation of guaiacol from enzymes become most optimal, and what has the highest reactivity rates. References Campbell, N and Reese, J. B (2006) Biology, p. 142-149, Pearson/Benjamin Cummings, San Francisco CA Marrs, K (2007) K101 Laboratory Manual, Ex. 5 “Characterization of Turnip Root Peroxidase” KhanAcademy (2012) “Oxidation and reduction cellular respiration” http://www. khanacademy. org/science/biology/cellular-respiration/v/oxidation-and-reduction-in-cellular-respiration.
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