i need a rough draft.

Hoffman Biology 1406 Lab

1

Lab Team Number: 2 Date: 10/13/2024

Team Members: Robert, Jay, Nesna, Nadia

Experimental Plan

1. After gathering and reading information about the topic, write down some questions or observations

that have surfaced.

Could the varied pH have a drastic effect on the absorbance even at a single increase of active acidity or alkaline base? At which pH could I record the least reaction to the bases control? Could some of the materials peroxidase concentration differ at a difference of sq/ft for each subject? What is the peroxidase concentration differ between sub species of cabbages and turnips?

2. Explain the purpose for standardizing the plant extract containing peroxidase (see the experiment).

We would be creating a standardized batch of the extract containing consistent concentration of peroxidase. It is important to have, to ensure reliable comparisons along with all the solutions used in the experiment. I will also help to determine the constant reactions of the peroxidase activity overtime and under varied pH conditions.

3. Write a null Ho and alternative Ha hypothesis for the affect of pH (see the experiment).

Null: The pH would have no significate effect on the activity and stability of the absorbance of the peroxidase, with no optimal pH range for activity or stability. Alternate: The pH significantly affects the activity and the stability of the cabbage and turnip peroxidase with a promising pH range for the peroxidase with the dye interaction changing the absorbance to 1 with minor margins from the literature.

Hoffman Biology 1406 Lab

2

4. From the affect of pH hypotheses: write a prediction for each plant tested (total of two). Predictions

must be about a specific pH your group will test or pH range. Do not make predictions about an

optimal pH or pH range.

Turnip: At a pH 6.0, peroxidase may present a decrease in the activity compared to the literature that was at pH 5.7, showing that it is less effective in a slightly acidic condition. Cabbage: At a pH 4.0 the cabbage peroxidase would retain substantial level of absorbance, through slightly lower than the optimal pH if 5.5, showing that it remains relatively active in the mildly acidic conditions.

5. Based on your team’s primary source findings, list the positive control pH buffer level your group will

use for each plant tested. When a decimal value is used (pH 5.5), round it.

Plant Positive Control pH buffer level Turnip pH 6.0

Cabbage pH 4.0

6. As support, list in the same order, citations from the selected primary sources (APA format).

Silva, M. C., Torres, J. A., Corrêa, A. D., Junqueira, A. M. B., Amorim, M. T. P., & dos Santos, C. D. (2012). Obtention of plant peroxidase and its potential for the decolorization of the reactive dye Remazol Turquoise G 133. Water Science and Technology, 65(4), 669–675. https://doi.org/10.2166/wst.2012.892 Joel, E. B., Mafulul, S. G., Adamu, H. E., Goje, L. J., Tijjani, H., Igunnu, A., & Malomo, S. O. (2020). Peroxidase from waste cabbage (Brassica oleracea capitata L.) exhibits the potential to biodegrade phenol and synthetic dyes from wastewater. Scientific African, 10, e00608-. https://doi.org/10.1016/j.sciaf.2020.e00608

7. Explain why the positive control pH buffer level provides an environment for an optimal peroxidase

reaction rate. In other words, explain how the positive control pH buffer affects peroxidase folding and

the shape of the active site.

The positive control pH buffer level is important for providing a prime environment of peroxidase activity

because of its effect on enzyme structure, mainly the folding and general shape of the active site. When

the enzyme is correctly folded while the active site is correctly shaped the reaction rate should increase.

A change in the pH level can change the chemical environment which is why positive control pH buffer

affects the shape and active folding.

Hoffman Biology 1406 Lab

3

8. List the experimental pH buffer levels for each plant tested. List them in the same order as in

statement five. Remember, the positive control pH level has been identified for each experiment.

Plant Experimental pH Buffer Levels Turnip pH 7.0 Cabbage pH 5.5

9. Explain why the experimental pH levels may not provide an environment that is optimal for

peroxidase reactions. As it relates to acidic and basic environments, use the terms protonation,

deprotonation and denaturing. In other words, explain how the experimental pH buffer levels affect

peroxidase folding and the shape of its active site.

If the conditions are acidic, having extra hydrogen ions can lead to protonation of some amino acid side chains. The changes or alterations could disrupt the ionic interactions that maintain the 3-D structure of the enzyme. When incorrectly protonation occurs the protein signals can destabilize, resulting in denaturing. While the proteins unfold in changes the active site shape, it can reduce the ability of the enzyme to bind to the substrate and catalyze a reaction. If the conditions are at a higher pH a protonated amino side chain can go through deprotonation. The change can effect the general charge distribution, basically disrupting the Hydrogen bonds and ionic interactions that stabilize the folded shape. Deprotonation can lead to misconfiguration of the active site, which could lead to the active site not being suitable for substrate binding.

10. State the independent and dependent variables of the experiment.

The independent variable is the time measured for the experiment. The dependent variable is the absorbance of the solution recorded from the experiment.

Hoffman Biology 1406 Lab

4

11. Qualitatively, explain how the rate of the peroxidase reaction will be measured. Include an

explanation of the chemical reaction.

The rate of the reaction can be measured by monitoring the absorbance of the solution at 470nm using the spectrophotometer, then recording the concentration and color change that increases in relation to the absorbance vs time. The ideal readings for the experiment would be a 1 absorbance at the end of the 2-minute mark. We are interested in the chemical reaction of the hydrogen peroxide buffer with the peroxidase and the final oxidated product. H2O2 + Guaiacol Dye + Peroxidase → 2H2O + Guaiacol Dye + Peroxidase (oxidized)

12. Quantitatively, explain how the rate of the peroxidase reaction will be measured. We will prepare all reaction mixture to their proper solutions mixture variants. We will start by recording the control first then record the changes of absorbance in the spectrophotometer at 470nm every 20 secs for a 2-minute span for each solution. Once the experiment is complete, we will calculate the rate of the reactions by determining absorbance overtime. A line graph will be used to find the slope of the absorbance vs time.

13. List the four parts of the experiment in chronological order.

1. Create the extract 2. Standardize the extract 3. Test the effect of the pH on extract 4. Graph – analyze