An Investigationin to the Effect of Ph on the Activity of Potato Tissue Catalase

An Investigation Into The Effect Of PH On The Activity Of Potato Tissue Catalase

Aim

The aim of my investigation is to see how pH affects the activity of potato tissue catalase, during the decomposition of hydrogen peroxide to produce water and oxygen.

Catalase + 2H2O2 Catalase + 2H2O +O2

Catalase + Hydrogen Peroxide Catalase + Water + Oxygen

Independent Variable

The independent variable in this investigation is pH. Each individual enzyme has it’s own pH characteristic. This is because the hydrogen and ionic bonds between вЂ"NH2 and вЂ"COOH groups of the polypeptides that make up the enzyme, fix the exact arrangement of the active site of an enzyme. It is crucial to be aware of how even small changes in the pH of an enzyme can affect activity until the enzyme will eventually become denatured.

The enzyme which is going to be used in this practical is catalase. Catalse is a very common enzyme which is found in almost all living and root vegetables such as, Celeriac and Potatoes. Catalase is a tetramer of four polypeptide chains, each over 500 amino acids long. It contains four porphyrin heme groups, which can explain why it suitable to catalyze hydrogen peroxide.

Dependant Variable

The dependant variable in my investigation is oxygen. There is a clear link between the independent and dependant variable. The enzyme catalase works at an optimum pH of 7. This means that the closer the pH is to 7, the faster oxygen will be produced. Also, as amount of substrate decreases, the rate at which the products are produced will also decrease. This is because there is a lot of substrate in the beginning of a reaction and therefore it is easier for the substrate to combine with the enzyme. But in time there will be more product than substrate which means it’s harder for the enzyme to combine with the substrate and as a result will take more time in combining with the substrate.

Hypothesis

In this practical, I predict that as the pH gets closer to pH 7, the oxygen will be produced more rapidly. This is because at a pH of 7 (neutral), the hydrogen and ionic bonds in the enzyme alter the shape of the active site, creating a suitable shape for the substrate to “fix” into the active site.

Preliminary Work

Before starting the practical, I had carried out a preliminary experiment. This was to observe the basic procedure I would be carrying out in the real experiment. This also gave me an idea of what results to expect in my practical and how I can perform a sensible experiment by being well aware of the corrosive acids and with the equipment being used. This also gave me an idea of how to keep my experiment fair as the preliminary experiment was done within two different room temperatures which gave an unforeseen result; which was completely different to the prediction I had made.

Controlled Factors

Temperature

It is vital to take the factors which can alter the rate of reaction into consideration. This includes temperature. When using catalase, temperature greatly affects the rate of a reaction because catalase has its own optimum temperature which is ordinarily exposed in it’s natural environment. Therefore, the closer the temperature is to the optimum temperature of catalase, the rate of reaction will rapidly increase until the enzyme will undergoe a procedure called denaturation; and then there will be a rapid loss of enzyme activity. To control this factor, the whole practical will take place in standard room temperature where the climate will be the same.

Concentration

Another factor which affects the rate of reaction is concentration. At low enzyme concentration there is great competition for the active sites and therefore the rate of reaction is low. But as the concentration increases there are more active sites to react with the substrate and therefore more oxygen gas will be produced until, the substrate becomes the limiting factor. To control the concentration I will take into consideration the availability of the enzyme molecules for the substrate and I will use the same amount throughout the practical.

Buffer Solutions

To prevent fluctuation in the pH, a solution known as a “buffer solution” was used in the experiment. Buffer solutions are mixtures of at least two chemicals which counteract the effect of acids and alkalis. Therefore, when a small quantity of alkali or acid solution is added the pH of the enzyme doesn’t change.

To make the buffer solution you need 0.2 mol dm-3 of Na2HPO4 and 0.1mol dm-3 of citric acid this will give 100cm3 of buffer. Here is how to get the different pH in the buffer solutions:

pH Na2HPO4 cm3 citric acid cm3

3 20.55 79.45

5 51.50 48.50

6 63.15 36.85

7 82.35 17.65

8 97.25 2.75

9

Apparatus

• Spotting tiles

• Stopwatch

• Three potatoes

• Cork borer

• Distilled water

• Beakers

• Buffer solution (5cm3)

• Hydrogen Peroxide (5cm3)

• Pipette

• Six boiling test tubes with rubber bungs

• Measuring Cylinder

• Manometer

• Stand, bosses and clamps

• Ruler

• Scalpel