The Effect of pH on the activity of enzyme Catalase

Variables:

Table 1:  Variables Selected for this Experiment

Materials and Methods:

1. Fresh potato
2. Boiling tube -1
3. Single bore rubber bung
4. Cork borer
5. White tile
6. knife
7. Small beaker
8. 2x10 cm3 measuring cylinder
9. Manometer tube (3mm in diameter)
10. Stop watch
11. Forceps
12. Ruler
13. 20 cc hydrogen peroxide
14. Range of buffers (pH-3-8)

Procedure:

1. Use a cork borer to cut cylinders of fresh potato tissue between  1 cm in length. Place on the tile to cut into at least 30   discs prepared for replicas for each sets each 1 mm wide.
2. Place all the discs in small beaker of water.
3. Clamp a boiling tube to a stand and carefully insert the manometer into rubber bung.
4. Using a syringe or small measuring cylinder place 5cm3 of buffer solution pH3 into the boiling tube.
5. Carefully add 10 pieces  of the potato discs followed by  5cm3  hydrogen peroxide solution.
6. Place the bung back into the boiling tube as quickly as possible.
7. Start  and stop watch and time  how long it takes for the manometer fluid to rise by  5 cm.
8. Carefully agitate the tube to make sure that the potato discs  do not  stick together.
9. Wash  out the boiling tube and repeat  the experiment using different pH buffer and ten new potato discs each time.
10. Make sure you use a clean syringe/measuring cylinder with each  different  buffer solution.
11. Each reading is repeated thrice for increased reliability.
12. One control set up was set up with 5 cc hydrogen peroxide and time was measured to increase the level of the manometer fluid by 5cm.

Data Collection and processing:

Raw data:

Results for control set up

Table 2

Data Collection and processing:

Raw data:

Table 3

Processed Table:

Table 4

Processed data:

Processed  Data:

Discussion, Evaluation and Conclusion:

This experiment supported the hypothesis.   It is evident that both high and low pHs inhibited the enzyme reactions. Enzymes are biological proteins that speed up the reaction rate of a chemical reaction. Enzyme activity depend on the temperature, ionic conditions, and pH of the surroundings.  The data showed that the enzymes had the best activity at pH 7. According to (Helms, Kosinski, and Cummings, 1998), at pH 7 the reactions will perform at an optimum level. This was proven true through this experiment we have conducted. As pH2 was not close to the optimum pH for the enzyme catalase, the reaction rate was slow and pH 9 may be a little high for activity to bind the substrate with the active site.  So we can see in these two pH that the amount of O2 production was not enough and time for the manometer fluid to raise 5 cm took more time.

There are a few problems in my investigations I have faced that need to be minimised for the next experiment. They are discussed as follows:

1.  Enzyme concentration of catalase varies with the age of the potato and even within the potatoes.
2. It was difficult for us to make the boiling tube air tight. Before the bung was inserted, there was a loss of gas from the test tube that could not be prevented.

These problems could be overcome quite easily for the next time this experiment is conducted.

To get the correct enzyme concentration, enzymes should be collected from the same part of the potato and there should be proper storage for the enzymes for at least two days prior to starting the experiment.

Bibliography

Brooker, Robert J et al , Biology , New York, Mc Graw-Hill 2008

Hershey, James et al. Biology 110 laboratory Text Book, New York, Pearson 2009  

Greenwood High School, Bangalore, India