Factors Affecting the Rate of Catalase Activity Essay

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Factors Affecting the Rate of Catalase Activity

Factors affecting the rate of catalase activity

The extent at which environmental factors affect the rate of catalase activity was discovered in this lab. The assay system, in which a filter paper disc was dipped into the enzyme and submerged using a stirring rod in a test tube filled with 20mL of hydrogen peroxide, was used to test several enzyme factors. As the saturation of hydrogen peroxide increased the rate of reaction increased as well. When the enzyme concentration increased the rate of catalase activity increased too. When catalase was subjected to an increase of temperature changes, the rate of reaction increased. Once the protein denatured around 100ºC the catalase activity decreased. Catalase operated with a high efficiency when the pH of the enzyme was 7. As the catalase pH was altered to acidic or basic, the rate of reaction decreased.

Introduction

Catalase is an enzyme that promotes the decomposition of hydrogen peroxide to water and oxygen gas: H2O2  H2O + O2. Hydrogen peroxide is a potential hazard for the body, which is why it is used to kill bacteria (WKU, 2010). Cells would die if hydrogen peroxide is not broken down by catalase. The activation energy for the decomposition of hydrogen peroxide is reduced from 72 to 20kcal/mol with the presence of catalase. Catalase is specifically found in the peroxisome, a membrane bound organelle (Xiaoling Li, 2002). Changing the concentration of the substrate and enzyme CiVf = CfVi(1) alters the environmental factors and affects catalase activity. When a hot plate and ice water were used to create water baths to change the temperature, the rate of reaction would be altered accordingly. 0.1 mol of NaOH and HCL were used to create an acidic or basic solution of catalase. Changing the pH of catalase would affect the expected enzyme activity.

Purpose

The purpose of this lab is to determine how environmental factors: pH, temperature, enzyme concentration, substrate concentration, and catalase activity in different tissues affect the rate of reaction

Question

How does changing the substrate concentration affect the activity of catalase? How does changing the enzyme concentration affect the activity of catalase? When the temperature is altered, how is the activity of catalase affected? If the pH of catalase is changed, how is the activity of catalase altered? How does catalase activity vary between tissues?

Hypothesis

When the substrate concentration is decreased, the enzyme activity will decrease accordingly. The rate of enzyme activity will amplify when the concentration of enzymes is increased. When the temperature is increased slightly, the rate of reaction will increase until the optimum value is reached and catalase is denatured. Catalase operates under a certain pH, the enzyme will denature when the pH is changed significantly.

Apparatus

– electronic balance
– 2 400mL beakers- 250mL erlymer flask – 100mL graduated cylinder – ring clamp – general clamp
– thermometer
– food processor
-stirring rod
– 4-6 25mL beakers
– forceps- plastic shot glass
-Petri dish – stop watch
– goggles
– hot plate
– cord stopper
– test tube holder
-insulated tongs
– 1L flask
– reaction chamber- 6-8 test tubes
– rubber tube- test tube rack- retort stand
– pen
-plastic ice container

Materials

– distilled water
– paper towel- pH paper
– paper disc
-tape – buffer 7
– HCl
– hydrogen peroxide 3%- red delicious apple
– 0.1 mol NaOH

– cheesecloth
– calf liver
-veal muscle
– golden potatoes- parafilm
– 0.1 mol HCL

Procedure

Part A: substrate concentration (assay technique)

1.add 20ml of 3% H2O2 to a test tube and measure depth
2.use tweezers to coat filter paper disc in the catalase enzyme 3.place the paper disc onto the stirring rod and submerge to the bottom of the test tube

1.add 15ml of 3% H2O2 and 5ml of H20 to a test tube to create a 2.25% solution 2.use tweezers to coat filter paper disc in the catalase enzyme 3.place the paper disc onto the stirring rod and submerge to the bottom of the test tube

1. Add 10ml of 3% H2O2 and 10ml of H20 to a test tube to create a 1.5% solution
2.use tweezers to coat filter paper disc in the catalase enzyme
3.place the paper disc onto the stirring rod and submerge to the bottom of the test tube

1. Add 5ml of 3% H2O2 and 15ml of H20 to a test tube to create a 0.75% solution
2.use tweezers to coat filter paper disc in the catalase enzyme
3.place the paper disc onto the stirring rod and submerge to the bottom of the test tube

1. Add 2.5 ml of 3% H2O2 and 17.5 ml of H20 to a test tube to create a 0.38 % solution
2. use tweezers to coat filter paper disc in the catalase enzyme
3.place the paper disc onto the stirring rod and submerge to the bottom of the test tube

1. Add 20 ml of H20 to a test tube to create a 0% substrate solution
2. use tweezers to coat filter paper disc in the catalase enzyme
3.place the paper disc onto the stirring rod and submerge to the bottom of the test tube

Part B: enzyme concentration (assay technique)

1.Add 20ml of 3% H2O2 to a test tube
2.use 5ml of 100u/mol of catalase
3.use tweezers to dip paper disc into catalase
4.place the paper disc onto the stirring rod and submerge to the bottom of the test tube

1.Add 20ml of 3% H2O2 to a test tube
2.dilute catalase 80u/mol: 4 ml 100u/mol catalase and 1ml of H20
3.use tweezers to dip paper disc into catalase
4.place the paper disc onto the stirring rod and submerge to the bottom of the test tube

1.Add 20ml of 3% H2O2 to a test tube
2.dilute catalase 50u/mol: 2.5 ml 100u/mol catalase and 2.5 ml of H20
3.use tweezers to dip paper disc into catalase
4.place the paper disc onto the stirring rod and submerge to the bottom of the test tube

1.Add 20ml of 3% H2O2 to a test tube and measure depth
2.dilute the catalase 20u/mol: 1ml 100u/mol catalase and
4 ml of H20 3.use tweezers to dip paper disc into catalase
4.place the paper disc onto the stirring rod and submerge to the bottom of the test tube

1.Add 20ml of 3% H2O2 to a test tube and measure depth
2.use tweezers to dip paper disc into H20
3.place the paper disc onto the stirring rod and submerge to the bottom of the test tube

Part C: Temperature (assay technique)

1.create a water bath of 0ºC, a room temperature bath 23ºC, 37ºC, and boiling water bath 100ºC 2.place 5ml of catalase at 100u/mol into 4 test tubes and set one in each water bath 3.allow test tube to sit for 5 min in the respective water baths 4.add 20ml of 3% hydrogen peroxide to 4 test tubes

5.used tweezers to dip 4 pieces of filtered paper discs into separate catalase baths 6.place disc on the bottom of the stirring rod and submerge each filter paper into a hydrogen peroxide test tube

Part D: pH (assay technique)

1.add 3% 20ml hydrogen peroxide to 3 separate test tubes
2.diluted catalase using 0.1 HCL and 0.1 mol NaOH to pH 1, 7, and 11.5 3.used tweezers to immerse a filter paper into each of the different catalase pHs 4.submerge the filter papers to the bottom of each respective test tubes

Part E: enzyme activity in different tissues (assay technique) 1.measured 20mL of 3% hydrogen peroxide into 4 separate test tubes 2.used tweezers to
immerse separate filter papers in muscle, liver, apple, and potato solutions 3.place filter paper onto stirring rod

4.submerge paper into hydrogen peroxide solution

Observations

A: Substrate concentration
substrate concentrationSubstrate depth (mL)Time (s)Rate (mL/s)pHEnzyme concentration (units/mol)Temperature (ºC) 3%201.0918.34710024
2.25%201.8710.70710024
1.5%201.9510.25710024
0.75%202.877.00710024
0.38%203.026.62710024
0.0%20N/AN/A710024
Figure 1: as substrate concentration decreases the rate of activity decreases

B: Enzyme concentration
Enzyme concentration (units/mol)Substrate depth (mL)Time (s)Rate (mL/s)pHSubstrate concentrationTemperature (ºC) 100201.3414.9373%24
80201.6612.0573%24
50201.7411.4973%24
20202.039.8573%24
020N/AN/A73%24
Figure 2: as enzyme concentration increases, catalase activity increases

C: Temperature
Temperature of catalase (ºC)Substrate depth (mL)Time (s)Rate (mL/s)pHSubstrate concentrationEnzyme concentration (units/mol) 100204.084.906.53%100
37201.5313.076.53%100
23201.6512.126.53%100
0201.4613.706.53%100
Figure 3: temperature increases until the optimal point is reached and catalase denatures

D: pH
pH of catalaseSubstrate depth (mL)Time (s)Rate (mL/s)Enzyme concentration (units/mol)Substrate concentrationTemperature (ºC)
1208.062.481003%22
7201.5512.91003%22
11.5203.166.331003%22
Figure 4: when the optimal pH of 7 is changed to acidic or basic, the enzyme denatures.

E: Tissue
Tissue typeSubstrate depth(mL)Time (s)Rate (mL/s)Enzyme concentration (units/mol)Substrate concentrationTemperature (ºC)pH
Apple206.553.051003%224
Potato2014.341.391003%226
Muscle205.833.431003%226
Liver201.3315.031003%226.5
Figure 5: catalase activity is greatest in liver and muscle, and weak in apple and potato

Discussion

1.If enzymes are reacting with substrates, and the substrate concentration is increased the activity of the enzyme will not be affected because they will already be occupied (Blake, 2002). On a graph with the rate of reaction on the y-axis, and the substrate concentration on the x-axis, once the saturation point is reached the graph will be horizontal because the rate of enzyme activity is not increasing. 2.The graph has a positive slope, as the enzyme concentration increases the rate of catalase activity increases. At the molecular level, as the concentration of catalase increases there are more enzymes to break down substrate. Subsequently the rate of enzyme activity will increase (Blake, 2002). 3.a. I) The greatest enzyme activity is indicated at 100 units/mol of catalase a. II) The lowest enzyme activity is indicated at 0 units/mol of catalase. b. as the enzyme concentration increases the rate of reaction increases because there are more enzymes to break down substrates.

4. As the temperature increased from 23 – 37 ºC the rate of catalase activity increased until the maximum on the graph at 37. However at 100ºC the catalase activity decreased significantly indicating catalase is denaturing. The optimal temperature for catalase should be around 37. 5.The pH value of catalase during the experiment was 6.5. 0.1 mol HCL and 0.1 mol NaOH were used to change the pH of catalase to acidic and basic values. These stock solutions enabled the testing of the activity of catalase at different pHs. 6. Catalase operates at an optimum pH of 7 (Worthington Biochemical Corporation, 2010). pH values that are too acidic or basic will denature the protein entirely by changing the active site.

7. Hydrogen peroxide is a toxic by-product from metabolic processes that produce free radicals (John Hewitson, 2010). Because the liver and muscle are more chemically involved in metabolic activity, a greater amount of hydrogen peroxide is produced and needs to be decomposed. Accordingly, the amount of catalase in liver and muscle is greater than that found in apple and potatoes. 8.Catalysts are substances that participate in a reaction to increase the speed at which it takes place it. Catalysts do not react with any of the reactants and are not used up chemically in the reaction. Not all catalysts are located in cells and can be made up of different elements. An enzyme is a protein that reduces the activation energy by breaking down substrates. Enzymes are used in cells to aid in metabolic processes.

All enzymes are catalysts abut not all catalysts need to be enzymes. 9.Activation energy is the amount of energy that is needed to initiate a chemical reaction. Organisms would produce enzymes to reduce the activation energy needed for reactions to take place. If the activation energy is lowered in a reaction, the amount of energy lost as heat is also reduced. In this way, when organisms produce enzymes like catalase not only will the reaction take place at a higher rate, but the reduction in activation energy will reduce the heat lost. 10. It would be expected to find an enzyme in humans at body temperature 37°C. If temperatures are too high or too low the enzyme will denature, changing its structure and rendering it useless. 11.To set-up the catalase solution, substances needed to be added which changed the pH to 6.5. In an in vivo test the optimal pH would be achieved but in vitro the pH is slightly altered. Furthermore, the temperature in vitro assays took place at was room temperature 22.5-24°C.

This differs from the in vivo temperature catalase operates at. Thus, in vitro tests slightly alter the optimal conditions at which catalase functions at 12.Homeostasis is the body’s ability to sustain certain environmental conditions (Blake, 2002). Carbonic anhydrase helps maintain homeostasis by converting hydrogen and bicarbonate ions to carbonic acid and vice versa. Hydrogen ions make the blood acidic, the body uses carbonic anhydrase to maintain this pH by increasing the rate at which hydrogen ions react with bicarbonate ions (Kimball, 2010). The human body also controls the activity of enzymes to maintain homeostasis (Starr C, 2010). When food enters the stomach, the hormone gastrin is released into the blood stream to trigger the release an amount of pepsinogen into the stomach to initiate the breakdown of proteins (Starr C, 2010).

The body controls pepsin in order to control and digest the protein in the stomach. 13.Using larger test tubes with greater amount of hydrogen peroxide will provide a greater reaction frame when timing how long the filter disc paper takes to travel to the surface. With 20mL of hydrogen peroxide, the time frame to stop and start the timer takes more precision. With a larger test tube more space is provided, and location of the filter paper as it travelling up the test tube is easier. In addition, instead of creating four hydrogen peroxide solutions and then conducting experiment, hydrogen peroxide solutions should be made only when needed. When hydrogen peroxide is exposed to light, the energy it received causes the initial decomposition of the solution. This affects the rate at which catalase decomposes the substrate as it had already begun the reaction.

Conclusion

Isolating variables, the environmental factors that affect the rate at which catalase decomposes hydrogen peroxide was examined. As the substrate concentration decreased from 3% and the volume remained consistent the rate of reaction decreased. According the collision theory, increasing the reactants increases the frequency of collision which increases the rate of the reaction (Nigel Purchon, 2001). When the concentration of catalase was decreased from 100 units/mol the rate of reaction decreased. The collision theory states, catalysts increase chemical reactions. Consequently, reducing the concentration of catalase decreases the rate of activity (Nigel Purchon, 2001).

The room temperature catalase was exposed to was 22.5 – 24 °C. As the temperature increased catalase activity increased until most of the protein denatured around 100°C. Once catalase denatured, the protein is rendered useless. The optimal pH of catalase, where the decomposition of hydrogen peroxide takes place at the highest rate, is 7 (Worthington Biochemical Corporation, 2010). When the enzyme was made acidic or basic the rate of reaction declined because the active site of catalase was altered. Lastly, catalase is more active in liver and muscle than apple and potato as more hydrogen peroxide is present in those tissues. The activity of catalase when the temperature was 0ºC was 13.70 mL/s. theoretically, catalase should denature at temperatures far enough from its optimal point.

The activity of catalase at this temperature is justified as the enzyme was allowed to gain heat once removed from the cold water bath. Contamination of lab equipment, for example the forceps, could lead to faster reaction times as traces if catalase from previous experiments may be present. The contamination lab equipment takes a toll on the theoretical results of the activity of catalase. Traces of catalase with different pHs, concentrations, or temperatures could be left on lab equipment. When contaminated lab equipment comes in contact with hydrogen peroxide, not all the variables are isolated and the results are changed. It is determined in vitro the environmental factors that affect the rate of catalase are substrate concentration, enzyme concentration, temperature, pH, and type of tissue.

References

Li Xiaoling, Stephen J. Gould (2002) PEX11 Promotes Peroxisome Division Independently of Peroxisome Metabolism. Retrieved from, http://www.bio.davidson.edu/Courses/Molbio/MolStudents/spring2002/Sellars/restricted/review.html Worthington Biochemical Corporation. (2010) Introduction to enzymes. Retrieved from, http://www.worthington-biochem.com/introbiochem/effectsph.html Purchon, Nigel. (2001). Rates of Reaction. Retrieved from, http://www.purchon.com/chemistry/rates.htm#concentration Hewitson John, Paul Beaumont. (2010) What affects the activity of the enzyme catalase. Retrieved from, http://www-saps.plantsci.cam.ac.uk/records/rec177.htm John W. Kimball. (October 6, 2010). Blood. Retrieved from,

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