The Oxidation Absorbency of Turnip Peroxidase by Hydrogen Peroxide (Containing Guaiacol) in Varying pH Levels

Janay Brecheisen, Tylynn Bourquin, Hao Nguyen, Chris Torrez, Ian Avila

IntroductionEnzymes are vital to metabolic processes in an organism. Their job is to lower the

activation energy needed for chemical reactions. If these reactions are too slow, organisms cannot function properly. Some factors that can affect the functioning of enzymes include substrate concentration, pH levels, and temperature. All enzymes have their optimal conditions at which they perform maximally. Outside of these conditions, enzymes can denature, or lose their shape, causing them to lose their function as they become incapable of matching with substrates.

In our experiment, we tested the functionality of turnip peroxidase, an enzyme that reacts with hydrogen peroxide to form oxygen and water. Make this a new sentence…in different pH solutions using a labquest and colorimeter (figure 1). We used guaiacol, a reducing agent, to indicate levels of oxygen released in the breakdown of hydrogen peroxide by the darkening shades of orange. What’s your hypothesis?

Procedure•We calibrated the colorimeter using a blank before beginning our experiment.•We put 1 mL of turnip peroxidase into a cuvette.•Next we placed 1 mL of pH solution into the cuvette.•We combined the turnip peroxidase and pH solution with 1 mL of hydrogen peroxide-guaiacol solution into the cuvette. (The two were not mixed simultaneously so they would not react until we were ready to begin recording data.) (figure 2)•We mixed the solution by turning the sealed cuvette upside down then right side up. We repeated this three times.•We placed the cuvette in the colorimeter.*As the reaction started, we pressed keep to record the absorbency at time zero. (figure 3)•We repeated steps four through six every thirty seconds for two minutes.•We completed this procedure for pH levels 3, 5, 7, 9, and 11 twice to have two sets of data for each of the pH levels.

Conclusion Each enzymatic reaction has an optimal

pH level where it functions at its highest rate. Changing the pH impacts the reaction rate. If the solution becomes too acidic or too basic the enzyme can denature and become non-functional.

In our investigation, we experienced an increase in the amount of light absorbance with the increasing pH levels. The treatments should have increased then decreased in absorbance level as the oxygen levels increased then decreased. The increase in absorbance is due to the darkening of the guaiacol as it binds to oxygen. The color change in our investigation was very slight, unlike what should have been observed. We had bubble formation in the cuvette, also.

Although we had excellent graphs, they were not a result of the color change in guaiacol. The increase in the absorbency levels was due instead, to an increase in the bubbles formed. The increase in bubbles decreases the available space that light can travel through; therefore, resulting in a higher absorbance level. The formation of bubbles could have resulted from the errors we experienced. We may have used a contaminated cuvette which could introduce unwanted material into the chemical reaction. Another possibility could be that the solutions were formed using a strong acid and a strong base. Both could result in a change in the concentration of the hydrogen and hydroxide ions in the solution also resulting in bubbles.

Recreating this lab would require conformation that each cuvette is brand new to eliminate the possibility of contamination. This experiment can be performed by college and high school students who have an understanding of the class and the equipment required. The way we conducted the experiment was not be the most reliable approach.

Base Run

00.05

0.10.15

0.20.25

0.30.35

1 2 3 4 5

Base Run

Time (sec) Absorbency 0 0.133

30 0.20560 0.25490 0.291

120 0.322

Trial 1

0

0.1

0.2

0.3

0.4

0.5

0 30 60 90 120

TIme (sec)

Abso

rben

cy

pH 11

pH 9

pH 7

pH 5

pH 3

00.05

0.10.15

0.20.25

0.30.35

0.40.45

0 30 60 90 120

Abso

rben

cy

Time (sec)

Trial 2

pH 11

pH 9

pH 7

pH 5

pH 3

Results

Figure 1

Figure 2

Figure 3