Enzyme Activity

Procedure 1: Develop a method for measuring peroxidase in plant material and determining a baseline(using water)

Materials:

Turnip Peroxidase
0.1% hydrogen peroxide
Guaiacol
Distilled(deionized) water
2 test tubes(approximately 16 x 150 mm) and appropriate test tube rack
Timer
1,5 and 10 mL graduated pipettes, pipette pumps, or syringes (1,2,5, and 10 mL)

Step 1: Mix 7.5 mL of distilled water, 0.1 percent hydrogen peroxide, and guaiacol in two test tubes, cover with parafilm, and gently mix

Step 2: Add distilled water and peroxidase to an enzyme tobe, cover with parafilm, and gently mix to create a total volume of 7.5 mL

Step 3: Mix substrate and enzyme tubes, cover with parafilm, invert twice, and place in test tube rack.

Step 4: Record color change at 0,1,2,3,4, and 5 minutes using a cell phone or camera, rotating the tube before each reading.

Step 5: Utilize the color palette/chart to measure color changes over time, and record your findings in your laboratory notebook

Procedure 2: Determining the effect of pH on enzymatic activity using buffers of various pH

Materials:

Turnip peroxidase
0.1% hydrogen peroxide
Guaiacol
Buffers with a range of pH 5, 6, 7, 8, 9
Distilled (deionized ) water
12 test tubes(approximately 16 x 150 mm) and appropriate test tube rack
Timer
1,5 and 10 mL graduated pipettes, pipette pumps, or syringes (1,2,5, and 10 mL)

Step 1: Create five sets of substrate and enzyme tubes using clean 16 x 150 mL test tubes. Substitute different pH buffers for distilled water in each pair. Add 7 mL of distilled water, 0.3 mL of hydrogen peroxide, and 0.2 mL of guaiacol to each substrate tube. Add 6.0mL of a specific pH solution and 1.5mL of peroxidase to each enzyme tube. Cover each tube with parafilm and gently mix

Step 2: Mix substrate tubes with enzyme tubes for five pairs, cover with parafilm, and gently mix. Place tubes back in test tube rack and start timing reaction

Step 3: Record the observed color for each tube at 0 munites and chosen time-based on procedure 1, using cell phones or cameras for color changes recording.

Step 4: Utilize a palette/color chart to measure observed changes, graph data in terms of substrate catalyzed/min versus pH, and draw conclusions from your findings.

Results/Analysis:

Discussions and Conclusions:

Claim: The presence of peroxidase in plant material leads to a visible color change when combined with hydrogen peroxide and guaiacol

Evidence: Starts with plant material suspected to contain peroxidase. The addition of hydrogen peroxide and guaiacol to this material follows. Peroxidase, if present, acts as a catalyst to speed up the reaction between hydrogen peroxide and guaiacol. This reaction resulted in a compound called tetraguaiacol, which has a brown color. Observation of color change in the mixture indicated the presence of peroxidase in the plant material. This color change serves as visual evidence supporting the initial claim. The speed of color change indicated the level of peroxidase activity. Faster color change means the reaction is happening more quickly, producing tetraguaiacol at a faster rate. In this experiment, the speed of color change provides additional evidence of peroxidase activity.

Reasoning: The claim and evidence support enzyme catalysis by demonstrating the role of peroxidase in speeding up the breakdown of hydrogen peroxide. Enzymes like peroxidase act as catalysts, which means they facilitate chemical reactions without being consumed in the process. In this case, peroxidase accelerates the decomposition of hydrogen peroxide into water and oxygen. Guaiacol is often used as a substrate in peroxidase experiments. When peroxidase comes into contact with guaiacol and hydrogen peroxide, it catalyzes the reaction that causes guaiacol to change color as shown in the chart of the different levels of color which represents the pH level. The evidence, such as the increased reaction rate and observable changes like color change, supports the idea that peroxidase enhances the efficiency of the reaction by lowering the activation energy required for the reaction to occur. This showcases the effectiveness of enzyme catalysis in biological processes.

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