Investigate the effect of temperature on the activity of catalase (from potato).

Aim: Investigate the effect of temperature on the activity of catalase (from potato).

Introduction: Enzymes are biological catalysts. They speed up metabolic reactions in the body but remain chemically unchanged themselves. Enzymes contain an active site. This is a region, normally a depression or cleft, to which another molecule may bind. This molecule is known as the substrate, and is usually specific to the active site of the particular enzyme, which breaks it down. Substrates will not usually fit into any other active sites other than that of the enzyme it is specified to. This can be explained as a lock and key model, where the lock and key are specific to each other, only, that there are many of the same kinds of lock and key when it come to the enzymes.

Just as lock and keys have three-dimensional shapes, proteins are also three-dimensional. Usually, there is only one active site on an enzyme; however there can be more. Some energy releasing reactions in cells produce hydrogen peroxide. This is acidic, and can thus, kill cells. Normally, hydrogen peroxide decomposes to form hydrogen and oxygen:

2H2O2 2H2O + O2

However, this process is very lengthy. There is an enzyme known as catalase in cells which dramatically increases the rate of decomposition of hydrogen peroxide.

catalase

2H2O2 2H2O + O2

This type of reaction where a molecule is broken down into smaller pieces is known as a catabolic reaction.

In order to investigate the effect of temperature on the activity of catalase, I will record the amount of oxygen released when hydrogen peroxide is broken down.

Variables: There are quite a few variables which can alter the rate of reaction, and need to be kept constant. They are as follows:

a) PH: at too high PH, the enzyme is denatured due to the loss of H+ ions. The same applies for too low a PH level, where too many H+ ions would attach to the negative regions of the enzyme, changes its shape and causing it to denature.

b) Concentration of enzyme: The higher the concentration, the higher the rate of reaction will be. With a larger number of catalase molecules, the chance of successful collisions between enzyme and substrate will be increased. In order to keep this constant, I will make sure I use the same volume of tissue (potato) containing catalase each time I conduct the experiment.

c) Surface area: The previous also applies to this.

d) Mass of tissue: Here it needs to be taken into account that different potatoes will not give the same mass, even if equally sized pieces are cut. Different potatoes will not have exactly the same water and catalase content.

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The mass will be kept constant in the same way as surface area and concentration of enzyme.

Prediction: The higher the temperature, the higher the rate of reaction up to a certain point. This is due to the fact that the particles gain kinetic energy and subsequently move around ...

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c) Surface area: The previous also applies to this.

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The mass will be kept constant in the same way as surface area and concentration of enzyme.

Prediction: The higher the temperature, the higher the rate of reaction up to a certain point. This is due to the fact that the particles gain kinetic energy and subsequently move around more vigorously. Thus, the chance of there being a successful collision between the enzyme and substrate molecule increases as reacting particles with collide more frequently with increased kinetic energy.

Enzymes have a very specific three-dimensional shape, held together by ionic and hydrogen bonds. If the amino acids are too vigorous in their motion, then, these bonds will brake. Once the bonds have been broken, the enzyme is said to have become denatured. As a result of becoming denatured, the enzymes' rate of activity becomes less because the enzyme loses its specific three-dimensional shape. The enzyme will start to become denatured after around 40?C as enzyme activity is usually at its optimum at this temperature. After this, the rate of reaction will probably deteriorate. After 60?C, there is likely to be no reaction, as the enzymes would probably be completely denatured by then. *(Above from: Source, Page 47)

Methodology:

1. Wear goggles for protection, and lab coat if available.

2. Arrange apparatus.

3. Get 250cm3 beaker and fill with water to about the 150cm3 mark with water at a specific temperature. This temperature can be reached by either cooling, via the use of ice, or by heating, via the use of a Bunsen burner.

4. While the water is reaching the desired temperature, get potato and bore out a cylinder with cork borer. To do this, place potato on tile and stabilise by gripping potato by the sides with one hand. Then, press down firmly with cork borer. To retrieve potato cylinder from within cork borer, push it out with the flat end of a pencil.

5. Cut the cylinder of potato obtained, with a ruler so that it is three centimetres long. Then, cut this three centimetre long cylinder up into a further six equal pieces, each 5 millimetres wide. Make sure to keep eyes level with ruler so as to minimise the chance of parallax error. The diameter of the cylinder remains constant due to the use of the cork borer.

6. Put the potato pieces into a boiling tube and put aside.

7. Measure out 30ml of hydrogen peroxide, using a 50cm3-measuring cylinder. Pour this into a boiling tube.

8. Get the two boiling tubes containing one containing hydrogen peroxide and the other potato. Put them both into the beaker of water once the water has reached the desired temperature (this can be done by heating or adding ice to water). After this, wait until the temperature of the hydrogen peroxide and the water bath are equal.

9. Once the temperatures of the potato and hydrogen peroxide are equal, clamp the boiling tube containing the hydrogen peroxide to a clamp stand. Then add the potato into the test tube

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containing the hydrogen peroxide and put bung on top of the test tube, which now contains both potato and hydrogen peroxide, and make sure it is in the beaker of water.

10. Now, measure the water being displaced from the 100cm3 measuring cylinder every thirty seconds using a stop watch.

11. Take readings every thirty seconds for five minutes.

12. Repeat the above steps for different temperatures.

Risk assessment:

During this investigation, I will use catalase from potato cells to speed up the breakdown of hydrogen peroxide. Hydrogen peroxide is an oxidising agent, as oxygen will be given off during the reaction. This implies that it could help the burning of fires. Due to this, reasonable care must be taken to ensure that the oxygen is not directly exposed to the flame of the Bunsen burner while water is being heated. As well as this, hydrogen peroxide can be very harmful if it enters the eyes. Thus, goggles must be worn. Also, if available lab coats will also be worn.

The cork borer is also a potential hazard as it is quite sharp. When cutting out the piece of potato, it must be ensured that the potato is resting on the tile before it is cut. The methodology explains how it will be cut.

The blade which will be used to cut the potato, is also another hazard. It must be handled with appropriate care as it is sharp.

Glass apparatus must be handled with care so that they do not break. If they do, that is also another hazard, and should be cleared up and disposed of in an appropriate fashion.

Fair testing:

While conducting the experiment, it has to be taken into account that a few variables will have to be controlled so as to ensure that the test is fair.

1. The PH of the reactants should be kept constant. This will not be hard to ensure as the PH of the reactants does not vary significantly during the course of the reaction.

2. The temperature of the reactants will have to be kept constant during the reaction. This is a variable which will be harder to control. There will be used, a beaker of 150cm3 of water at the appropriate temperature into which will be immersed, the boiling tube containing the reactants once their temperatures have equalised and stabilised. This will ensure that the temperature of the reactants is stable for longer as the larger volume of water in the beaker will maintain its temperature for longer. Also, the same volume of water will be added to the beaker each time a new test is to be done, so as not have any differences in how long the water maintains its heat for.

3. It must be ensured that when taking readings off the measuring cylinders and thermometers etc, the readings are taken with the graduated markings at eye level, so as to minimise the risk of inaccurate readings.

4. All boiling tubes etc. must be washed thoroughly with water after their use, so as to minimise the chance of contamination.

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5. Making sure that all the potato pieces are cut to the same width, and have the same diameter will control the surface area of the potato.

While conducting the experiment, it must be ensured that the utmost attention is paid to accuracy due to the fact that accurate measurements will result in accurate results. Subsequently, the evidence for or against my prediction regarding the enzyme activity in relation to temperature will be more strong, and, more reliable. The above mentioned methods of accurate measurement will be used as they are most appropriate for the situation, with time factors being one of the major reasons for the augmentation of such methods, as well as the availability of equipment.

During the experiment, the following apparatus will be used:

Cork borer (size four)

Potato

Hydrogen peroxide

Water

Blade

Ruler (15cm)

Stop watch

Test tube rack

Boiling tubes

Delivery tube and rubber bung

Water container

Measuring cylinders (100 cm3 and 50cm3 )

Goggles

Cutting tile

Clamp and stand

Beaker (250cm3)

Graduated pipette (5ml)

ice

Thermometer (0 ?C-100 ?C)

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During the experiment, the apparatus will be set up in the way shown below:

During preliminary work, I found that the 100 cm3 measuring cylinder was ideal for the measurement of water being displaced by oxygen formed during the reaction. This is because it is graduated in millilitres. Thus, it will be possible to measure the displacement of water to the nearest 0.5 of a millilitre. Also, the measuring cylinder is not so small that the volume of oxygen produced will be greater than the cylinder can hold within the course of the reaction.

The 50cm3 measuring cylinder used to measure the volume of hydrogen peroxide was ideal as the amount of hydrogen peroxide was wasn't too little that it would be inappropriate and also inaccurate to measure with a 50cm3 measuring cylinder, nor was it so large, that it would just about be measured.

Boiling tubes are ideal for the reaction to take place in, as the volume of oxygen produced is quite small. Thus, it will be quicker for the oxygen produced to be able to displace the water in the measuring cylinder. With a conical flask, it would take much longer.

I also found that the breakdown of hydrogen peroxide at room temperature is very slow (Without a catalyst). No oxygen was given off at all over the period of time I observed the hydrogen peroxide for any reaction. However, I did not test whether this was true for higher temperatures. If it was not, then there is the likelihood of major inaccuracies in the conducting of the experiment.

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Source:

Background information used during this investigation was obtained from Cambridge Advanced Sciences. Biology 1 - endorsed by OCR: Chapter 3. (Page 42 onwards.)

Also, information on variables was taken from concepts learned in AS chemistry: (Salters Horners Advanced Chemistry)