Catechol Oxidase Lab Essay

Catechol oxidase is an enzyme that speeds up the oxidation reaction when catechol is exposed to oxygen. When the reaction occurs, benzoquinone is produced turning the oxidized substance brown. It was hypothesized that the higher the concentration of catechol oxidase, the browner the substance will turn, and the faster it will achieve the color. In the present lab, different concentrations of catechol oxidase were mixed with pure catechol and the rate at which each solution browned was measured using a colorimeter.

The results showed that the solution with the high concentration of catechol oxidase had the fastest rate at which it turned brown. However, it did not turn the brownest, the solution with the medium concentration of enzyme did. These results show that our hypothesis was both supported and not supported and it was concluded that increase in catechol oxidase concentration increases rate of turning brown but does not increase the level of brownness a substance becomes.

Introduction

Have you ever wondered why the inside of your apple turns brown if you leave it on the counter after taking a bite? This is because there is a compound within the apple called catechol which, when exposed to the air, becomes oxidized by the oxygen in the air and produces benzoquinone, which makes the apple appear brown (Andrew Clapper’s Online Journal, 2007). This chemical reaction is catalyzed by the enzyme catechol oxidase (Figure A.1). Enzymes are proteins in cells that speed up chemical reactions (Enzyme Procedure Guide, 2013). The catechol oxidase lowers the amount of energy needed in order for the catechol to be oxidized, which speeds the process up.

Fig. A.1: The oxidation process of catechol with the help of catechol oxidase produces benzoquinone. (Enzyme Procedure PowerPoint, 2013)

If you frequently eat apples you may have also noticed that different types apples brown at different rates. This is because each kind of apple has a different concentration of catechol oxidase (Daniela Finkel, 2013). Therefore, each apple has a unique speed at which it will produce benzoquinone, and turn brown.

This experiment was designed to investigate the following hypothesis: The higher the concentration of catechol oxidase, the browner the substance will be, and the faster it will achieve the color (Enzyme Procedure Guide, 2013).

Within the experiment, pure catechol was mixed with different concentrations of catechol oxidase and the rate at which each solution produced benzoquinone was measured. The amount of benzoquinone made throughout the trials was measured by using a colorimeter to measure the level of “brownness” of the liquid. The colorimeter worked by shining a light through the liquid and then measuring that light on the other side to see how much of it was absorbed.

In this experiment, absorbance of blue light was measured because blue light is absorbed by the color brown. The amount of blue light absorbance was measured every 15 seconds for five minutes. Because enzymes speed up reactions, more enzymes would cause the reaction to be even faster.1

Procedure/Materials and Methods

The lab leaders and the Punk Rock Warlord prepared three different concentrations of catechol oxidase by extracting potato juice (because it contains lots of catechol oxidase). Pure catechol, a 5mL test tube, 1mL/5mL syringes, a colorimeter and water were also used. 4mL of pure catechol and 1mL of the highly concentrated catechol oxidase were measured with the syringes. Both liquids were then injected into the 5mL test tube. The 5mL tube was placed into the opening in the colorimeter and the lid of the colorimeter was closed.

The measure of absorbance of blue light displayed on the screen of the colorimeter was recorded every 15 seconds for five minutes. This procedure was repeated for two more trials, each with 4mL of pure catechol; one used 1mL of medium concentrated catechol oxidase and one used 1mL of low concentrated catechol oxidase.

The independent variable was the concentration of catechol oxidase, the dependent variable was the rate of turning brown, there were no controls, there were three replications and the sample sizes were 11 (for high concentration) and 10 (for medium and low concentrations).

Results Section

Figure 1 shows that before the rates began to plateau, the high enzyme concentration solution was turning brown the fastest. Table 1 shows that the high enzyme concentration had the fastest rate and that on average, it deviated from the mean more than the medium and low concentrations. Figure 2 shows that the high enzyme concentration solution was turning brown the fastest. Table 2 shows that the different rates each solution turned brown are statistically significant.

Table 1: Average Rates of for High, Medium and Low Concentrations of Catechol Oxidase and the Absolute Average Deviations. Three different concentrations of catechol oxidase were added to pure catechol and the rate of change in the absorption of blue light (production of benzoquinone) was measured every 15 seconds using a colorimeter. Data shown as mean of % of absorption per minute. N=11 for the high concentration and n=10 for the medium and low concentrations.

Concentration of Catechol

• Average Slope (Rate)

Abs. Ave. Dev.

• High

1. 0.818
2. 0.174

• Medium

1. 0.569
2. 0.081

• Low

1. 0.196
2. 0.120

Table 2: T-Test Results When Comparing the Rates at Which Different Solutions Browned Three different concentrations of catechol oxidase were added to pure catechol and the rate of change in the absorption of blue light (because production of benzoquinone makes a substance brown) was measured every 15 seconds using a colorimeter. N=11 for high concentration and n=10 for medium and low concentrations.

Enzyme Concentrations

P-Value
Low vs. Medium