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Enzymes Essay

Abstract
The enzyme that is responsible for the darkening of cut surfaces of fruits, vegetables and plants are called polyphenoloxidase. These enzymes, like all all other biological catalysts that cause us to exist, are often taken for granted. Without this enzyme, fruits, plants, and vegetables would-be left unprotected from different infections and diseases. There would be no response to the injuries, tissues in plants, fruits and vegetables might incur. Also, an absence polyphenoloxidase would leave humans without a skin pigment to tan. Thus, to understand this enzyme more in depth this paper will show the results of the research done on the effects of different concentrations of the enzyme, and the effects temperature will have on the rate of the reaction. In theory, the reaction rate should be proportional to enzyme concentration (“Factors Affecting Enzymes”); thus, the outcome of the experiment was successful. The results for the effects on temperature also appeared to be consistent with the hypothesis that reactions take place best in 35° C; temperatures close to normal body temperature.

Introduction
Have you ever wondered what causes the darkening of cut surfaces of fruits, vegetables and plants? First, to understand the process of this phenomena, we must understand how enzymes, the biological catalysts work. The process of an enzyme can be very complex. “The enzyme will catalyze the reaction by binding to a substrate molecule and altering its molecular structure so that the substrate is more readily converted to a different molecule or product” (Campbell 96, 97)
Astoundingly, the enzyme that is responsible for the darkening of cut surfaces of fruits, vegetables and plants are called polyphenoloxidase. “Polyphenoloxidase catalyzes the oxidation of a catechol to ortho-quinone and then undergoes a series of changes to form a red product” (Koning).

The reaction is:
cathecol+ Ѕ O2 Polyphenoloxidase ortho-quinone + H2O red product
Thus, the result of the reaction is a response to injury, the catechol is released and the enzyme is converted to ortho-quinone, which is an antiseptic to the injured tissue. So the brownish effect of the cut surface protects the plant from infection or disease (Koning). Moreover, the enzyme polyphenoloxidase can also be found in humans by a different name of tyrosinase, which produces skin pigment melanin, which causes tanning.

Thus, the objective of the trials that will be done on the enzyme polyphenoloxidase is to witness the effects of different concentrations of the enzyme, and effects the temperature will have on the rate of the reaction. The theory is rate of the enzyme reaction should be proportional to the enzyme concentration (“Factors Affecting Enzymes”). Also, the reactions of the temperature should react best in 35° C due to the closeness to normal body temperature.

Materials and Methods
Effect on Enzyme Concentration
The procedure to find out the effect of the polyphenoloxidase enzyme concentration and effects on temperature on polyphenoloxidase began, October 4, 2002 and continued on October 11, 2002. The first step of the experiment was to prepare the enzyme, polyphenoloxidase, by washing and peeling a potato. It was then important to chop it into pieces and blend it with 40 ml of phosphate buffer for 1 to 3 minutes. The blending caused the tissues of the potato to homogenize. After the potato and the phosphate buffer were blended, the solution was then strained into a test-tube through two layers of cheesecloth in a funnel. Amazingly, the initial color of the filtered enzyme immediately changed from a cream color to a light brown as soon as it was poured into the test tube. The final steps of the preparation of the potato enzyme involved the filtration in the centrifuge for five minutes for the removal of cell wall, cell fragments and starch grains. The solution was then poured into a test-tube and then placed in a beaker of ice to keep the enzyme cold.

During the preparation of the polyphenoloxidase enzyme, the materials that were going to be used in the experiment were prepared, such as one clean empty test-tube, one Spec. tube, and one Spec. tube half filled with the phosphate buffer. A very interesting instrument was used and it was called the Spec.20 Spectrophotometer; it is used for measuring the transmission of light by comparing various wavelengths. It was vital that the Spec.20 Spectrophotometer was set to 520 nm and set to zero, before the experiment began. The Spec. tube that was half filled with the phosphate buffer was used to set the Spec.20 Spectrophotometer to zero. The final step that was done before the trial test took place was the preparation of the substrate. The preparation of the substrate involved the mixing of 10 ml of 0.006 cathecol solution with 40 ml of the phosphate buffer in a beaker.

Finally, all necessary steps were taken to begin the trial test of the experiment. Using a pipette, 10.0 ml of the substrate was mixed with 0.4 ml of the enzyme extract, and .6 ml of the phosphate buffer in a test-tube. The test-tube was immediately transferred to the Spec.20 Spectrophotometer and the stop watch was started. There was a Spec. reading every minute for 10 minutes. After 10 minutes, the test-tube was taken out, shook briefly and put back in the Spec.20 Spectrophotometer for several more minutes. The trial was completed and the Spec. readings were taken.

Next, the real trial was preformed, known as trial one. This time there was a control group. In the control .5 ml of the enzyme and 5.5 ml of buffer were mixed together. In another test-tube the solutions that were mixed included: .5 ml of enzyme, .5 ml of buffer, and 5 ml of buffer-substrate. The control test-tube was first put in the Spec.20 Spectrophotometer and only one control reading was taken. The control was immediately taken out and the trial test-tube was quickly put in. Spec. readings were taken every minute for 10 minutes. After the Spec. readings of the test-tube trial, the control reading was again put in the Spec.20 Spectrophotometer for one final control reading.

Following the first trial, the second trial began. The second trial involved the combination of: .8 ml of enzyme, .2 ml of buffer, 5 ml of buffer-substrate. The control trial involved .8 ml of enzyme and 5.2 ml of buffer. The control test-tube was first put in the Spec.20 Spectrophotometer and only one control reading was taken. The control was immediately taken out and the trial test-tube was quickly put in. The Spec. readings were taken every minute for 10 minutes. After the Spec. readings of the test-tube trial, the control reading was again put in the Spec.20 Spectrophotometer for one final control reading.

Lastly, for the third trial for the effect of enzyme concentration, 1 ml of enzyme,
5 ml of buffer-substrate were combined. For the control group 1 ml of enzyme and 5 ml of buffer were mixed. The control test tube was first put in the Spec.20 Spectrophotometer and only one control reading was taken. The control was immediately taken out and the trial test-tube was quickly put in. The Spec. readings were taken every minute for 10 minutes. After the Spec. readings of the test-tube trial, the control reading was again put in the Spec.20 Spectrophotometer for one final control reading.

After all of the trials were completed, the results were plotted on a same piece of graph paper. To find the initial rate of the enzyme concentration, a straight line was drawn through as many points that could form a straight line. Thus, the initial rate was found in the slope of the straight line.

Temperature Effect
Following the experiment of the effect of enzyme concentration, another procedure was done, to see the temperature effects on the polyphenoloxidase enzyme. The procedure involved the preparation of the enzyme as described before. Then Spec.20 Spectrophotometer was set up the same way as in the previous experiment. As in the past experiment, the buffer-substrate was also prepared. However, in each of the four test- tubes that would be tested, 3 ml of buffer, and a required amount of enzyme would be added. So a trial test was done by adding 5 ml of buffer-substrate, and .5 ml of the enzyme. It was then placed in the Spec.20 Spectrophotometer and readings were made every minute for five minutes and thus the required amount of enzyme was established. Finally, by establishing required amount of enzyme, .5 ml of enzyme and 3 ml of buffer were poured into four different test-tubes. The test-tube that was the control was kept at room temperature, the second test-tube was placed boiling water for four minutes and then cooled under the water at room temperature. The third test-tube was boiled at 35 0C, and then cooled as well. The fourth test- tube was placed in a beaker of ice. The experiment began with the placing of each test tube one after the other in the Spec.20 Spectrophotometer in sequential order for 10 minutes while also noting the readings.

Results
Enzyme Concentration Reaction
The preparation of the phosphate buffer, cathecol and phenolxidase yielded a promising outcome. After much preparation, quick moving and contemplation on the experiment which involved four trials the outcome was graphed, as seen in the graph of The Effect of Enzyme Concentration. Then the initial rate was figured out and graphed as seen in the graph of Initial Rate of Enzyme Concentration. A certain pattern was noticed in the effect the substrate had on the enzyme concentration. In the test trial, 10.0 ml of the substrate was mixed with 0.4 ml of the enzyme extract, and .6 ml of the phosphate buffer in a test-tube. The initial rate of this enzyme concentration turned out to be .10608 Spec. per min. During the very first trial, in the control .5 ml of the enzyme and 5.5 ml of buffer were mixed together. In another test-tube the solutions that were mixed included: .5 ml of enzyme, .5 ml of buffer, and 5 ml of buffer-substrate. The initial rate of this enzyme concentration turned out to be .11325 Spec. per min.

The second trial involved the combination of: .8 ml of enzyme, .2 ml of buffer,
5 ml of buffer-substrate. The control trial involved .8 ml of enzyme and 5.2 ml of buffer. The initial rate of this enzyme concentration turned out to be .11825 Spec. per min. Finally, in the third trial for the effect of enzyme concentration, 1 ml of enzyme, 5 ml of buffer-substrate were combined. For the control group 1ml of enzyme and 5 ml of buffer were mixed. The initial rate of this enzyme concentration happened to be .2997 Spec. per min.

Effect of Temperature Rate of Reaction
In the second part of the experiment, how the phenolxidase would be affected in different temperatures was the primary focus. The outcome of each treated tubes as compared with the control was varied and can seen in the graph of Temperature Effects. Compared to the control, the test tube that was placed in boiling water denatured the enzyme and caused its relative rate of reaction to stay the same. The test-tube that was 35 C compared to the control had the best relative rate of reaction since it is pretty close to the normal body temperature. The test-tube that stayed in the ice-bath, had very little activity in the ice bath due to the enzymes and substrate moving at a very slow speed so there was not much interaction. Finally, the control itself that was at room temperature moved at a moderate speed. Thus, it was shown that significance of each temperature effect was very important; because it increased the reaction of polyphenoloxidase, decreased the reaction, or denatured the enzyme.

Discussion
The objective of this experiment was to find the effects of different concentrations, and temperatures on enzyme reactions. In theory, the reaction rate should be proportional to enzyme concentration (“Factors Affecting Enzymes “). Thus, during each concentration trial the absorbance should have increased with time like it had in the entire trial test and the other three trials.

First of all, errors and problems could have occurred early in the experiment during the preparation of the enzyme when there might have been an accidental dropping of some mixture while decanting the potato. Other problems included the fast pace of the experiment, the solution for the trials had to be made very quickly especially when it concerned the transfer of the enzyme. Last but not least, the major error in the experiment had occurred within the Spectrophotometer, when it broke down in the middle of trial 3. The test tube had to be immediately transferred to another Spectrophotometer, and continued from there.

The second part of the experiment was based on the different temperature effects of enzyme reactions. As based on the hypotheses, the test-tube that was 35 0C had the best relative rate of reaction since the reaction took place close to normal body temperature. The test-tube that stayed in the ice-bath, had very little activity in the ice bath due to the enzymes and substrate moving at a very slow speed so there was not much interaction as expected. The test tube that was placed in boiling water at 100° C denatured the enzyme and caused its relative rate of reaction to stay the same. Thus, it was shown that significance of each temperature effect was very important; because it increased the reaction of polyphenoloxidase, decreased the reaction, or denatured the enzyme. Thus, the enzymes’ optimal temperature is around body temperature, when most reactions occur.

Errors that could have occurred with this experiment seem to be very minimal, since part of the experiment was a repeat of the previous trials that were done on the effect of different enzyme concentrations. An error could have been made with finding the required amount of enzyme to use. Errors could have taken place during the Spec. readings, when each of the four tubes were sequentially being put in the Spec. and taken out after the reading. This was especially frustrating when everything was going on at such a fast pace that there could have definitely been a mix-up of the tubes with different temperatures.

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