Peroxidase lab report PDF

Preview

Summary

peroxidase lab report...

Description

Effects of Temperature, pH, and the Enzyme Inhibitor Hydroxylamine on Peroxidase Enzymes extracted from Brassica rupa subsp. rapa.

Abstract: The purpose of this lab is to investigate the impact temperature, substrate concentration, enzyme concentration, pH and the presence of the competitive inhibitor hydroxylamine on the effectiveness and reaction rate of the enzyme peroxidase. We obtained the peroxidase enzyme from the Brassica rupa subsp. rapa. The enzyme activity was measured using the absorbance of each solution along with the conditions being tested. The temperatures effect on enzyme activity was tested using the the different temperatures; 4 degrees Celsius 23, 38, and 47. Each temperature was measured in intervals of 20 seconds from 20 to 120. The pH variable was tested using the same steps as the temperature, the pH that were tested were pH 3, pH 5, pH 7, and pH 9 every 20 seconds until 120 seconds had elapsed. After collecting the data from each tested variable, temperature, pH level, and the presence of Hydroxylamine. We deduced that as the temperature of the enzyme’s environment is increased, then the rate of the reaction will also increase. If the substrate concentration is increased, then the rate of the reaction will increase. As the environment of the enzyme’s pH changes, so does the reaction rate. When testing for the optimal pH for enzyme activity, we found that at the low pH 3 and the maximum pH 9 enzyme activity was significantly decreased. While testing both pH 5 and 7 we observed an increase in enzyme activity. The change is enzyme activity based on our findings leads us to believe that the optimal pH for enzyme activity of peroxidase is within the pH range of 5-7. The final variable we tested was the change in enzyme reaction when the inhibitor (hydroxylamine) is added to the peroxidase enzyme. After adding hydroxylamine to the peroxidase solution the enzyme reactions decreased significantly, to the point that the enzyme become denatured. In addition to adding

hydroxylamine to the peroxidase one set of the solution was boiled while the other was not. Although the decrease did not denature the enzyme, it only dramatically decreased the reaction rate. then the rate of the reaction will decrease significantly.

Introduction: Enzymes are proteins that carry out chemical reactions, and are catalysts within living organisms that regulate the rate at which a chemical reaction occurs. Enzymes range in their functions throughout living organisms. For example metabolic enzymes are found in all cells of the body, because they are essential in the breakdown of lipids, carbohydrates, proteins and other molecules in the cell. The enzymes within the human body are amylase which breaks down starch into sugar, protease which breaks down proteins into amino acids, lastly lipase which breaks down lipids into fatty acids and glycerol. (Pūtaiao) The general function of all enzymes is to act as a catalyst, chemical agents that increases the rate of a chemical reaction without itself undergoing any permanent chemical change. (Dolphin) Each enzyme has an active site, part of the molecule that has just the right shape and functional groups to bind to one of the reacting molecules. The reacting molecule that binds to the enzyme is called the substrate. An enzymecatalysed form a reaction intermediate. This formation has a lower activation energy than the reaction between reactants without a catalyst. The enzyme is used to form a reaction intermediate, but when this reacts with another reactant the enzyme reforms.(rsc.org) The reaction catalyzed by peroxidase is the oxidation-reduction decomposition reaction of hydrogen peroxide. Before performing our experiment to investigate the impact temperature, substrate concentration, enzyme concentration, pH and the presence of the competitive inhibitor hydroxylamine on the effectiveness and reaction rate of peroxidase hypotheses were made. The

rate of an enzyme-catalyzed reaction in regards to temperature should increase as the temperature increases. Enzymatic adversely affected by high temperatures because as the heat increases so does the reaction until it hits the maximum then the reaction begins to decline. If pH is related to enzyme activity, then changing pH will result in major activity change. If the enzyme is not at its optimal pH, when at a very high or very low pH in comparison to the enzymes’ optimal pH the result will be complete loss of activity. The final variable, the inhibitor hydroxylamine will cause a decrease in the reaction rate of the enzyme. The definition of an inhibitor is a molecule that binds to an enzyme and decreases its activity. The inhibitor binds with the active site of an enzyme blocking the substrate from binding which results in a denatured enzyme.

Materials: ● Constant temperature water baths

● 5ml pipettes

● Spectrophotometers

● 10 mM H₂O₂

● Blender

● 25 mM guaiacol

● Fresh turnip

● 2% Hydroxylamine

● Markers

● Citrate-phosphate buffers at pHs 3,5,

● Spectrophotometer cuvettes ● 10 ml test tubes and rack ● 50 ml beakers

Methods:

7, and 9 ● Peroxidase (turnip)

Set up nine sample tubes to observe how enzyme reactions work and to examine the effects of enzyme concentration on the rate of activity. Remember when using a spectrophotometer, you must first zero the instrument using a blank each time you test a new tube. Prepare the blank and the required number of experimental tubes. Use labeling tape and a marker label each of the tubes. Make sure the experimental tubes are well mixed once. Follow the procedure in your lab book to test each variable every 20 seconds for 3 minutes you will measure absorption, using 470 nm as the wavelength. Record the absorbance of each sample tube in Tables 6.1-6.10 in the lab book. (Dolphin, W.D. (2015) Determining the properties of an enzyme, Biological Investigations: Lab Topic 6 pp. 69-78). After conducting all of your experiments use the results from your tables to construct a graph of enzyme rate based on each variable (temperature, pH, hydroxylamine inhibitor, boiled peroxidase). Discussion: The peroxidase was tested at 4 Degrees Celsius and had a slower reaction than when tested at 23 Degrees and 38 Degrees. This occurred as extreme low or high temperatures slow reaction rate as the environment is not suitable for the peroxidase enzyme. Then, when the temperature was increased to 23 and 38 Degrees Celsius, the rate of reaction became more intense, as the environment was favorable. However once the temperature got too high at 47 Degrees Celsius, the reaction began to slow down at an extreme rate. Very high and very low temperatures slowed the reaction rate as all enzymes can only withstand temperatures within their natural environment. When the peroxidase was tested with different pHs, a pH of 5 gave the enzyme the highest reaction rate. Just as with temperature, a too high or too low pH will throw off the enzyme’s rate or reaction as all different enzymes have a specific pH they can endure to have the

best reaction. At a pH of 3 for instance, the reaction rate barely changed at all, supporting the fact that enzymes prefer a more neutral pH rather than very acidic or basic. When the peroxidase was boiled, the reaction was almost unchanged as the enzyme was denatured. However, unboiled peroxidase thrives as it is more suitable for the enzyme. The hydroxylamine was used as an inhibitor for the enzyme, causing it to stop the reaction rate and again destroy the enzyme. This non-competitive inhibitor binds to the enzyme through weak non-covalent interactions, stopping the reaction from occurring usually as it should. In the future, testing different inhibitors would be interesting to see whether the enzyme would react the same or differently. Perhaps competitive inhibitors react differently than non-competitive inhibitors.

Citations: Brassica rupa subsp. rapa. (n.d.). Retrieved October 2019, from https://www.cabi.org/isc/datasheet/10118.

Dolphin, W.D. (2015) Determining the properties of an enzyme, Biological Investigations: Lab Topic 6 pp. 69-78

“Enzymes.” Chemistry for Biologists: Enzymes, 2015, https://www.rsc.org/Education/Teachers/Resources/cfb/enzymes.htm

Science Learning Hub – Pokapū Akoranga Pūtaiao. (2011). Digestive Enzymes. https://www.sciencelearn.org.nz/resources/1840-digestive-enzymes...