Factors that Affect Catalase activity

Bernadette Walsh

February 2008

Factors that Affect Catalase activity:

Introduction:

The aim of this experiment is to find out how the rate of a typical enzyme-catalysed reaction varies with different factors. In this experiment, enzymes will be investigated by using Catalase. A certain factor will be chosen and altered, in order to observe the effect on the activity of the enzyme.

The main roles of enzymes are to act as biological catalysts in order to generate movement, assist active transport, and are involved in the digestive systems of animals; breaking down macromolecules into smaller ones.

Background Information:

Enzymes

An enzyme is a biological catalyst, which is found in all living organisms. All enzymes are globular proteins made of long chains of amino acids, which fold to form specific tertiary structures. The tertiary structure of an enzyme determines its function.

¹“Enzymes are large molecules that work by reacting with another compound (the substrate) to form a short-lived enzyme-substrate complex. This complex is formed at a particular part of the enzyme molecule, usually at its surface, known as the active site. The complex breaks down to form the product(s), leaving an uncharged enzyme molecule, which is then available to catalyse another cycle. At the active site the enzyme works by reducing the amount of energy required (activation energy) which substrate molecules need to have before they can undergo the chemical change concerned.”

There are two models, which explain enzyme activity, the lock-and-key model, and the induced-fit model. Please see diagrams of each in Fig 1 below.

2: Fig 1.

The lock-and-key model basically shows a temporary attachment between the active site and the substrate. The substrate must be an exact fit with the active site for the reaction to occur.

The basis for the induced-fit model is that the enzyme provides an alternative reaction pathway with a lower activation energy. After the enzyme binds to the substrate, the enzyme may change shape. This is due to stress being put on to internal chemical bonds, such as hydrogen bonds of the enzyme. This then allows the reaction to take place under lower energy requirements.

Enzymes may be denatured by a number of factors such as temperature and the PH value. Other factors such as the concentration of the enzyme, concentration of the substrate, the source of enzyme, the surface area, volume, and inhibitors will also affect the activity of the enzyme. This will be explained further in the ‘Factors’ section.

Enzymes contain hydrogen bonds, which hold the enzyme in its specific 3D shape and help enzymes bind to their substrate. The hydrogen bonds can easily be broken by the factors mentioned above. When the hydrogen bonds are broken, the shape of the active site of the enzyme is changed. As a result, enzyme-substrate complexes can no longer be formed; therefore inhibiting any further reaction.

Activation energy:

Activation energy is the energy required for a reaction to take place. The lower the activation energy of a reaction, the faster the reaction and vice versa. An enzyme reduces the activation energy. See graph below.

5Energy Against Reaction Path:

When an enzyme binds with a substrate the available energy has a greater effect and the rate of catalysis increases.

Catalase:

3 “Catalase is a common enzyme found in root vegetables, such as potatoes and celeriac. It catalyses the decomposition of hydrogen peroxide to water and oxygen.” This reaction mainly takes place in a cellular organelle called the Peroxisome. Peroxisomes are found in both animal cells and also in plant cells.

3 “Peroxisomes in plant cells are involved in photorespiration (the use of oxygen and production of carbon dioxide) and symbiotic nitrogen fixation (the breaking apart of diatomic nitrogen (N2) to reactive nitrogen atoms). All known animals use catalase in every organ, with particularly high concentrations occurring in the liver.”

The reaction is as follows:

2H₂O₂ → 2H₂O + O₂

3 “Catalase is a tetramer of four polypeptide chains, each over 500 amino acids long. It contains four heme (Iron) groups that allow the enzyme to react with the hydrogen peroxide. The optimum PH for Catalase is approximately PH 7.0.”

Factors:

Temperature:

Temperature is a measurement of heat of a solid, liquid or gas, measured in degrees Celsius or Kelvin.

To investigate how the temperature affects Catalase activity, I will use a water bath to vary the temperatures of the enzymes. The rate of the reaction is expected to increase with increasing temperatures, exhibiting a proportional relationship. It is also expected that the rate will increase with a directly proportional relationship until a certain point (the optimum point), after which the rate should decrease due to the denaturation of the enzymes present.

Higher temperatures will contain molecules with ...

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Factors:

Temperature:

Temperature is a measurement of heat of a solid, liquid or gas, measured in degrees Celsius or Kelvin.

Higher temperatures will contain molecules with a higher average kinetic energy. This will produce a higher number of successful collisions, with energy sufficient to surpass the activation energy, resulting in a faster reaction rate.

4: Reaction Rate against Temperature:

It relates to the practical in the way that temperature can be changed to alter Catalase activity.

pH:

The pH of a substance shows how strong or weak it is with respect to the hydrogen ion concentration, and is measured on a scale from 0-14.

To investigate how the pH affects Catalase activity, I will use hydrogen peroxide solutions of different pH’s. A bell curve will be expected, when a graph of pH is plotted against reaction rate. This is because at lower pH’s, the enzymes will be denatured due to the acidity of the solution, and at very high pH’s, the enzyme will be denatured due to the alkaline solution. The reaction rate should be highest at a particular point, which will be its optimum pH.

4: Reaction Rate against pH:

It relates to the practical in the way that pH can be changed to alter Catalase activity.

Concentration of enzyme:

The concentration of enzyme is the amount of enzymes contained in the certain substance.

To investigate this factor, I would carry out the experiment several times with different amounts of potato (source of Catalase) each time. I would start with a low amount and gradually increase the amount each time.

As the enzyme concentration increases, the more active sites there are available for substrate molecules. This will continue until there is an excess of enzyme molecules for the amount of substrate molecules. After this point, increasing enzyme concentration will have no effect; the substrate concentration becomes a limiting factor.

4: Reaction Rate against Enzyme Concentration:

It relates to the practical in the way that the concentration of the enzyme can be changed to alter Catalase activity.

Concentration of substrate:

The concentration of substrate is the amount of substrate contained in the certain substance.

To investigate this factor, I would carry out the experiment several times with different amounts of hydrogen peroxide (substrate) each time. I would start with a low amount and gradually increase the amount each time.

As the substrate concentration increases, more enzyme-substrate complexes are formed. This will continue until there is an excess of substrate molecules for the amount of enzyme molecules, as the active sites will be saturated. After this point, increasing substrate concentration will have no effect; the enzyme concentration becomes a limiting factor.

4: Reaction Rate against substrate concentration:

It relates to the practical in the way that the concentration of the substrate can be changed to alter Catalase activity.

Surface area:

The surface area of an object is a measure of how much of its area is exposed and is measured in square units.

To investigate this factor, I would carry out the experiment several times with potatoes of the same volume, but different surface areas. I would start with a small surface area (i.e. potato in one piece), and then I would repeat the experiment, each time cutting it into more pieces (i.e. into two, then into 3 and then into 4 pieces etc.).

The larger the surface area, the faster the reaction rate will be. This is because with a larger surface area, substances can diffuse through a lot faster and easier than if there was a small surface area.

It relates to the practical in the way that the surface area of the potato can be changed to alter Catalase activity.

Choice of Factor:

I have chosen temperature as the factor I will be experimenting because it allows you to choose a suitable range for the values of controlled or independent variable. The dependent variable will be the amount of oxygen produced. All the other variables will be fixed.

Independent variables:

The amount of oxygen gas produced.

Controlled variables:

Temperature; 15˚C, 25˚C, 35˚C, 45˚C, 55˚C, 65˚C and 75˚C
Time test tube is in water bath
Reaction time (time to produce oxygen gas).

Prediction:

Hypothesis: As temperature increases, the amount of oxygen produced increases, until the optimum temperature is exceeded, when which the enzyme gets denatured.

Reasoning: As temperature increases, the speed of movement of the substrate and enzyme molecules increases. This is because with higher temperatures, the enzymes and substrate obtain more energy and therefore can move faster, increasing the number of collisions between the substrate and active sites of the enzymes. The optimum temperature, which is the temperature enzymes work best at will be reached. After the optimum temperature is reached, the structure of the enzyme molecule starts vibrating so energetically that some of the bonds (e.g. hydrogen bonds) holding the enzyme molecule together in its precise shape begin to break. The enzyme molecule’s active site therefore starts to lose its shape and once this happens, will no longer be able to bind with the substrate (enzyme is denatured).

Predicted Observations: The amount of oxygen produced will increase with increasing temperature. Once the optimum temperature for the enzyme has been reached, the amount of oxygen produced will stop increasing. As temperature continues to increase after the optimum temperature, the amount of oxygen will decrease, as after this point the enzyme will be denatured.

Time and Temperature Factors: I will ensure these factors are controlled and will stay the same by using a thermometer and a stopwatch. I will time the amount of time the test tube is in the water bath and also the amount of time the hydrogen peroxide is in the test tube, to keep it constant throughout the experiment. The temperatures will be chosen before the experiment and will be constantly measured to ensure the water baths are at the right temperature.

Apparatus:

The Preliminary Method:

1. Set up apparatus as shown below.

2. Using a cork borer and razor blade, cut 5 cylindrical sections of 2cm of potato.

3. Place one section of 2cm of potato into a test tube.

4. Half fill a beaker with water (to be used as a water bath) and heat/cool down, using a Bunsen burner/ice to get a temperature of 10˚C.

5. Place the test tube in a water bath at 10˚C, as shown below.

6. Ensure water bath is at correct temperature and manometer and syringe are securely in the test tube.

7. Mark a starting position on the manometer, where the flouracine ends (on the side away from the Bunsen burner, to avoid any accidents which may occur).

8. Add 2ml of hydrogen peroxide to the test tube, using a syringe.

9. Start the stopwatch and time for one minute.

10. After one minute, mark the ending position on the manometer.

11. Using a ruler, measure the amount of oxygen produced by measuring the distance between the two points on the manometer.

12. Repeat steps 2 – 11, only using 15˚C, 20˚C, 25˚C, 30˚C, 35˚C, 40˚C 45˚C, 50˚C, 55˚C, 60˚C, 65˚C and 70˚C water baths.

13. Clean test tubes.

14. Repeat steps 2 – 13 twice.

Safety Precautions:

Preliminary Results:

The results show that the highest amount of O₂ produced is 12.6 cm³. A denaturing point is not shown and therefore an optimum temperature cannot be worked out.

Preliminary Conclusion:

This experiment did not support my hypothesis, as temperature increases, the amount of oxygen produced increases, until the optimum temperature is exceeded, when which the enzyme gets denatured.

Evaluation:

Firstly, my experiment did not match my hypothisis which shows there was certainly something wrong with the method. The amount of hydrogen peroxide was insufficient for the amount of potato (i.e the end of the potato was above the surface of the hydrogen peroxide. This shows that there should have been more hydrogen peroxide and less potato or shorted disks of potato.

The markings on the manometer would have only been approximate since it is fairly difficult to mark, therefore the ammount of oxygen produced may have been read/noted incorrectly. This would have cause inaccuracy where the amount of oxygen produced is concerned. Also, extra pressure from placing the syringe into the testube will have changed the point at which the flouracine begins and ends in the manometer and therefore again would result in innaccuracy.

Finally, some time would have been taken to start and stop the stopwatch. Therefore, the lengths of time will not have been exactly the same for all of them.

Changes to make to preliminary experiment:

Hydrogen peroxide:

Due to the fact that there was an insufficient amount hydrogen peroxide for the potato, I have decided a change from 2ml of hydrogen peroxide to 5ml of hydrogen peroxide is necessary.

Lengths of Potato:

Due to the fact that the potato was above the surface of the hydrogen peroxide, I have decided that the lengths of potato should be changed from 5cm to 2cm, and cut into four pieces of 0.5 cm so a larger surface area is available.

Climatisation:

Due to the fact that the enzymes did not have enough time to change temperature, I have decided to increase the length of time the test tube is in the water bath from 1 minute to 3 minutes.

Reaction time:

The length of reaction time will increase; the hydrogen peroxide will be left in the test tube for 3 minutes instead of 1 minute.

Temperatures:

Higher temperatures to be tested, since an optimum temperature and denaturing point were not observed.

Apparatus:

Only 7 test tubes required instead of 13 test tubes.

Final method:

1. Set up apparatus as shown below.

2. Using a cork borer and razor blade, cut 4 cylindrical sections of 0.5 cm of potato.

3. Place the 4 sections of potato into a test tube.

4. Half fill a beaker with water (to be used as a water bath) and heat/cool down, using a Bunsen burner/ice to get a temperature of 10˚C.

5. Place the test tube in a water bath at 15˚C, as shown below.

6. Leave the test tube in the water bath for 3 minutes.

7. Ensure water bath is at correct temperature and manometer and syringe are securely in the test tube.

8. Mark a starting position on the manometer, where the flouracine ends (on the side away from the Bunsen burner, to avoid any accidents which may occur).

9. Add 5ml of hydrogen peroxide to the test tube, using a syringe.

10. Start the stopwatch and time for three minutes.

11. After three minutes, mark the ending position on the manometer.

12. Using a ruler, measure the amount of oxygen produced by measuring the distance between the two points on the manometer.

13. Repeat steps 2 – 12, only using 25˚C, 35˚C, 45˚C, 55˚C, 65˚C and 75˚C water baths.

14. Clean test tubes.

15. Repeat steps 2 – 14 twice.

Bibliography: