Enzyme Activity: Experiment Study Of Peroxidase

Abstract

An enzyme is a biological catalyst. The catalyst studied in the experiment was peroxidase. The source of peroxidase used in this experiment was from turnip extract. Guaiacol was used in this experiment to be able to tell how much light was absorbed by the brown color the test tube turned from the guaiacol. We examined the amount of light absorbance with this enzyme with a different treatment. A spectrophotometer was used to measure the amount of light absorbance. We doubled the amount of turnip extract which allowed for more light absorbance. We used half the amount turnip extract which allowed for less light absorbance. We changed the pH of the solution from neutral to a pH of 3 and a pH of 11. Both pH changes killed the reaction enzyme allowing for no light absorbance. We also used an analogue in a test which allowed for no absorbance as well. The analogue confused the enzyme which caused it to bind with the analogue instead of the substrate causing no absorbance to occur. Lastly, we used boiling turnip extract instead of regular turnip extract. The boiled turnip extract allowed for less light absorbance because the heating of the enzyme denatured it causing for less of a reaction. Overall, the results show that the amount of light absorbance is affected by different treatments on the peroxidase enzyme.

Introduction

Most reactions in cells proceed with the help of a catalyst. A catalyst works by lowering the activation that is required for the reaction to get started. This is how the reaction is sped up. Enzymes are usually large proteins made up of a bunch of amino acids. Some enzymes however contain a non-amino acid as well which is called a cofactor. A cofactor can be simple or complex organic compound. If it is an organic molecule it is considered a coenzyme. Since an enzyme is a protein it has a three-dimensional structure that is held together by strong forces. An enzyme has an active site that binds to a substrate. The product is produced when the binding happens. When the product is produced, the enzyme goes to bind to another site and repeats.

In this experiment, we tested how the enzymatic reaction changes in response to different treatments being added to the original solution. We measured a baseline solution to use as the original to compare our treated solutions to. There was also a baseline solution that was also used as a blank to clear the spectrophotometer. I expected that when the concentration of the enzyme was doubled, the amount of light absorbance would increase. I expected that when the concentration of the enzyme was cut in half, the amount of light absorbance would decrease. I expected that when the amount of pH was greater, that the absorbance would be lower, and I expected that when the amount of pH was lower, the absorbance would be higher. I expected when the analogue was added that there would be no light absorbance at all. I expected that when the boiled enzyme was used that the light absorbance would decrease because the enzyme would be denatured because of the heat.

Methods

First, we made the baseline solution tube. The baseline tube consisted of 0.1 mL guaiacol, 1.0 mL turnip extract, and 7.9 mL of water. We then put the baseline in the spectrophotometer and adjusted the absorbance to zero. Next, we created the original solution that we compared the treatment tubes to. The original tube consisted of a mixture of two tubes. The first tube contained 0.1 mL guaiacol, 0.2 mL H2O2, and 3.7 mL of water. The second tube contained 1.0 mL turnip extract and 4.0 mL of water. We mixed the two tubes together and immediately placed it in the spectrophotometer. We recorded the amount of absorbance every 30 seconds for 3 minutes. Then we created the first treatment tube. The first treatment tube was the double enzyme concentration. This test also consisted of two tubes. The first tube contained 0.1 mL guaiacol, 0.2 mL of H2O2, and 3.7 mL of water. The second tube contained 2.0 mL of turnip extract and 3.0 mL of water. We mixed the tubes like before and placed it in the spectrophotometer and recorded the absorbance again every 30 seconds for 3 minutes. The second treatment tube was the half enzyme concentration. The first tube contained 0.1 mL guaiacol, 0.2 mL of H2O2, and 3.7 mL of water. The second tube contained .5 mL of turnip extract and 4.5 mL of water. We mixed the tubes and recorded the absorbance every 30 seconds for 3 minutes. The third treatment tube was the pH 3 treatment. The first tube contained 0.1 mL guaiacol, 0.2 mL H2O2, and 3.2 mL of pH 3 solution. The second tube contained 1.0 mL of turnip extract and 4.0 mL of pH 3. We mixed the tubes and recorded the absorbance every 30 seconds for 3 minutes. The fourth treatment tube was the pH 11 treatment. The first tube contained 0.1 mL guaiacol, 0.2 mL H2O2, and 3.2 mL of pH 11 solution. The second tube contained 1.0 mL of turnip extract and 4.0 mL of pH 11. We mixed the tubes and recorded the absorbance every 30 seconds for 3 minutes. The fifth treatment was the analogue treatment. The first tube contained 0.1 mL guaiacol, 0.2 mL H2O2, and 3.7 mL of water. The second tube was prepared differently than usual. First, 1.0 mL of turnip extract was placed in the tube. Then, 10 drops of 10% hydroxylamine solution was placed in the tube. We let the tube set for 2 minutes then added 4.0 mL of water. We mixed the tubes and recorded the absorbance every 30 seconds for 3 minutes. The last treatment boiled the enzyme treatment. The first tube contained 0.1 mL guaiacol, 0.2 mL H2O2, and 3.7 mL of water. The second tube contained 1.0 mL of boiled turnip extract and 4.0 mL of water. We mixed the tubes and recorded the absorbance every 30 seconds for 3 minutes. After all the treatment tests, we had all the data for the lab.

Results

Treatment one (double enzyme concentration) had more light absorbance than the original test. Treatment two (half enzyme concentration) had less light absorbance than the original test. Treatment three (pH 3) had no light absorbance because the high pH killed the enzyme. Treatment four (pH 11) had no light absorbance because the low pH killed the enzyme. Treatment five (analogue) also had no light absorbance since the enzyme mistook the analogue for the substrate and bound to it instead. Treatment six (boiled enzyme) had less light absorbance because the heat-denatured the enzyme causing less of a reaction.

Discussion

My first hypothesis was supported. When the concentration of the enzyme was doubled, the amount of absorbance increased. My second hypothesis was also supported. When the concentration of the enzyme was cut in half, the amount of absorbance decreased. My third hypothesis was not supported. When the amount of pH was higher, there was no absorbance, and when the amount of pH was lower, there was no absorbance. My fourth hypothesis was supported. When the analogue was added, there was no light absorbance. My fifth hypothesis was also correct. When the boiled enzyme was added, the amount of absorbance decreased.

When the concentration doubled, the absorbance increased because there was more enzyme to react. When the concentration was cut in half, there was less absorbance because there was less enzyme to react. When the pH was added, both low and high, there was no light absorbance because the non-neutral pH level killed the enzyme reaction. When the analogue was added, there was no absorbance because the enzyme got confused and binded to the analogue instead of the substrate. When the boiled enzyme was used, the absorbance was less because the heat-denatured the enzyme which caused it to not work like it was supposed to.

References

1. Mbuthia, K. W. (7th Edition) Concepts in Biology II Laboratory Manual. Cincinnati, OH: Van-Greiner Learning