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Enzymes Lab Report

principles of biological science i laboratory (bsc 110l ), university of southern mississippi.

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Experiment 1: Determining How Enzymes Work as a Catalyst BSC 110L Section H Nadia Peyton TA: Helen WeberOctober 28, 2019

The experiment was constructed to determine how enzymes would react when mixed with pH buffers. Enzymes are catalyst which speed up the rate of reaction. I believed that pH 10 would have the greatest reaction because it was the highest pH buffer. When doing this experiment, I used hydrogen peroxide, guaiacol, and deionized water. I mixed a certain measurement of each one of these together, and some blanks had pH buffers added to them. I then measured the changes every 15 seconds for 5 minutes. The results determined that the higher the pH buffer the greater the reaction rate will be. In my discussion, I stated that my hypothesis was correct because enzymes can work alone, but it had some help which made it work faster. This experiment showed how enzymes can work as a catalyst and speed up the rate of reaction with pH buffer 10.

INTRODUCTION Enzymes have always been an important part of science. They are proteins, that can act as catalyst, that are within living organisms, and they carry out chemical reactions (Castro). Enzymes help break large molecules into smaller pieces, and this helps the body absorb them more easily. They are also highly selective catalyst which means that each enzyme speeds up a specific reaction (Castro). There are many different types of enzymes, but they function according to their amino acid that make up the protein. Without enzymes in the cell, the reaction would take for too long to be carried out. Enzymes speed up reactions by lower the activation energy (Boncompagni).

measured 2g of turnip, which is a source of peroxidase, and blended the turnip with 150ml of deionized water. I had to filter the mixture to ensure that there are no chunks. After blending my mixture, I had to prepare my blank. To do this, I needed to fill a cuvette with 8 of deionized water, 0 of guaiacol, and 1ml turnip, which is my enzyme solution. After I made the blank, I waited a couple of seconds to ensure that nothing happened because nothing was supposed to happen. I sat that aside to calibrate my colorimeter and making sure that I knew how to properly use it. I then took a bigger testing tube to mix 8 of deionized water, 0 of guaiacol, and 0 of hydrogen peroxide. No reaction should happen with this mixture. I continued to put a piece of Dura-Film over the test tube to shake everything together. After my mixture was mixed together well, I filled my cuvette about 2/3 full and put it in the colorimeter. I then timed the colorimeter readings every 15 seconds for 5 minutes. I was then assigned to be a base, so I didn’t have to do anything for part 2 of the experiment. In part 2, I was supposed to put a pH buffer in place of the deionized water, but I mentioned before I did not have to participate in this part.

RESULTS AND ANALYSIS In this experiment, there were a variety of different results. This lab experiment determined that high pH buffers makes enzymes react faster. The graph demonstrates the relationship between the rate of the reaction and the pH buffer. As the concentration of the pH increased, so did the rate of the reaction. The data clearly presents the effect of a pH buffer and how it significantly sped up the rate of the reaction.

The graph is found on below and it allows for further conclusions to be drawn.

0 0 15 30 45 60 75 90 105 120 135 150 165 180 195 210 225 240 255 270 285 300

The rate of enzyme activity with pH

BasepH 4. pH 7 10.

Figure 1: This line graph shows how the enzyme changed when it had pH mixed in with it. The number 0-300 represent the seconds, and 1-6 represents the numbers that were received while watching the colorimeter.

DISCUSSION When an enzyme solution is mixed with a pH buffer, the highest buffer will cause the fast rate of reaction. This hypothesis was supported by my graphed data because pH buffer 10 had the highest results. I was instructed to fill out the base column, and that column rarely changed. Every other experiment that included pH buffers had significant results. Their numbers changed drastically over the course of 15 seconds, so that leads me to believe that my hypothesis was supported by simple science. Enzymes won’t change unless it has something strong enough to help it do so. This experiment had little to no

LITERATURE CITED

Castro, Joseph. “How do Enzymes Work?” LiveScience, Purch, 26 April 2014, livescience/45145-how-do-enzymes-work.html Arnold, Ben. “The Working Principles of Colorimeters.” AZOSensors, 8 August 2019, azosensors/article.aspx?ArticleID= Boundless, Biology. “Enzymes.” Lumen, 2016, courses.lumenlearning/boundless- biology/chapter/enzymes/. Brennan, John. “How to Use Lab Pipettes.” Sciencing, 24 Apr. 2017, sciencing/use- Boncompagni, Tatiana. “Enzymes Try to Grab the Spotlight.” The New York Times, 22 lab-pipettes-8391951. Feb. 2012, nytimes/2012/02/23/fashion/enzymes-once- sidelined-try-to-grab-the-spotlight

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Course : Principles of Biological Science I Laboratory (BSC 110L )

University : university of southern mississippi.

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Enzyme Reactions: Discussion and Results

Table 1. Solution concentrations, volumes and observations for Experiment 1: Observing the enzyme reaction.

*The chemical reaction was observed with the introduction of catechol to the potato extract (tube 2).

Table 2. Solution concentrations, volumes and observations for Experiment 2: The effect of substrate concentration on enzyme activity.

*The reaction time for tube 1 was the fastest due to the high substrate concentration and lower dH 2 0 concentration.

Table 3. Solution concentrations, volumes and observations for Experiment 3: The effect of enzyme concentration on enzyme activity.

*Lower Dh20 and higher potato extract concentrations allowed for a faster reaction time

for tube 1.

Table 4. Solution concentrations, Buffer pH, volumes, and observations for Experiment 4: The effect of pH on enzyme activity.

*The reaction rate increased as the pH increased, with a pH of 6 being the best buffer for catechol oxidase activity. Increasing the pH past 6 showed a decrease in the reaction rate.

Table 5. Solution concentrations, temperatures, volumes and observations for Experiment 5: The effect of temperature on enzyme activity.

*The fastest reaction rate was observed at 37°C. The colder the temperature (0°C – 15°C), the slower the reaction rate. Enzyme denaturation was observed at 100°C.

Table 6. Solution concentrations, volumes and observations for Experiment 6: Inhibitor Effects – Inhibiting the Action of Catechol Oxidase

*The fastest, and most pronounced reaction was observed in tube 1 (the solution without phenylthiourea)

Enzyme Lab Discussion

For the first experiment, Observing the Enzyme Reaction, it was hypothesized that the enzyme reaction would only occur in the second test tube due to the fact that it was the only tube to contain both the enzyme and substrate. As expected, the solution in tube 2 was the only solution to show the characteristic yellow-brown pigment of benzoquinone production, which was caused by the potato extract converting its catechol into the new product.

In experiment 2, The Effect of Substrate Concentration on Enzyme activity, the hypothesis was that the tube with the higher substrate concentration would show a faster and more pronounced chemical reaction than the tube with less catechol.

The hypothesis was supported by the fact that the higher catechol concentration in tube 1 allowed for a similar result to tube 2 from experiment 1, the only difference being that the extra 5mL of dH 2 0 diluted some of the yellowish-brown color observed in the first reaction.

While there was a chemical reaction observed in tube 2 (experiment 2), it was much slower (with a translucent peach pigment) due to lower a catechol concentration and a higher dH 2 0 concentration. The higher the concentration of catechol, the more benzoquinone that can be produced.

It was hypothesized in experiment 3, The Effect of Enzyme Concentration on Enzyme Activity, that the higher the concentration of enzyme in the solution, the faster and more pronounced the chemical reaction would be.

This hypothesis was able to be accepted based on the rate at which the tube with the higher potato extract concentration reacted. Tube 1 had 400μL more potato extract and 400μL less dH 2 0 than tube 2. Because enzymes are biological catalysts that speed up chemical reaction time, the solution in tube 1 quickly changed from a bluish-green pigment, to the yellowish-brown color associated with benzoquinone.

The lower concentration of potato extract and a higher concentration of dH20 in tube 2 showed no change in color, other than the cloudiness of the potato extract itself.

In experiment 4, The Effect of pH on Enzyme Activity, the initial hypothesis was that the lower the pH level of the buffer added to the solution, the quicker the reaction rate would be. This hypothesis was not supported by the data observed because higher acidity levels actually slowed the production of benzoquinone – which was the opposite of what was predicted.

The solution with a pH buffer of 4 remained cloudy white, while the solution with a 6 pH buffer turned yellowish-brown. As the pH increased, the benzoquinone production rate increased. While lower pH buffers proved to be too acidic, more neutral buffers allowed for the best environment for catechol oxidase activity.

Buffer pH levels higher than 6 showed a slower and less pronounced chemical reaction as well – illustrating the enzyme reaction’s need for neutrality.

The hypothesis for experiment 5, The Effect on Temperature on Enzyme Activity, was that extremely low temperature would slow the rate of benzoquinone production, while extremely high temperatures would cause the enzymes to denature. This hypothesis was supported by the rate at which the solutions at 0°C – 15°C slowly reacted, and the rate at which the solution at 37°C quickly produced benzoquinone.

After five minutes at each solution’s designated temperature, the colder solutions barely started to change color, while the warmer temperatures quickly reacted – so much so that at 100°C, the enzymes denatured and the solution began to pale in pigment. Colder temperatures slowed the movement of molecules in the solutions, while warmer temperatures (not including 100°C) allowed for a better environment for catechol oxidase activity.

For experiment 6, Inhibitor Effects – Inhibiting the Action of Catechol Oxidase, it was hypothesized that the addition of phenylthiourea (PTU) would keep the enzyme reaction from occurring. The hypothesis was able to be accepted due to the fact that the tubes which contained the PTU showed very little change in pigment.

Tube 1 served as the control, which showed the production of benzoquinone (yellowish-brown color) and allowed for comparison between the three solutions. Considering PTU is a non-competitive inhibitor, tubes 2 and 3 contained solutions that prevented the enzyme from catalyzing the reaction, regardless of whether or not the substrate was bound to the active site.

The only real issue with any of the 6 experiments was the unsupported hypothesis for the Effect of pH on the Enzyme Activity experiment. I must have tied the preservative nature of benzoquinone with how acidic lemon juice keeps apples from turning brown, so I assumed a low pH would increase the reaction rate. In reality, acidity slows the reaction rate – which is why the apples don’t change color.

In conclusion, these experiments have shown that benzoquinone production can only occur with the presence of both an enzyme and substrate. Factors such as substrate and enzyme concentration, pH, temperature, and the presence of noncompetitive inhibitors can affect enzyme reaction. High substrate concentration will allow for greater benzoquinone production, while high enzyme concentration will speed up the reaction rate – and vise versa.

In order for enzyme reaction to rapidly occur, it must be done in an environment where the pH is as close to neutral as possible, with the reaction rate slowing in both highly acidic or basic solutions. The same goes for temperature – extremely high or extremely cold temperatures can decrease enzyme reaction rates, or cause the enzymes to denature altogether.

The introduction of a noncompetitive inhibitor (such as phenylthiourea) allows it to bind to the allosteric site on the enzyme, which keeps the reaction from occurring (regardless of the enzyme or substrate concentration).