The Effect of pH on Catalase

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The Effect of pH on Catalase

Hypothesis:  The rate at which catalase catalyses the decomposition of hydrogen peroxide increases to an optimum pH and then decreases as the optimum pH is exceeded.

        

Biological knowledge:

        Hydrogen peroxide is produced in plant and animal cells as a by-product, which is toxic, resulting in the need of catalase to speed up the decomposition of hydrogen peroxide to water and oxygen.  All microbodies contain catalase and it is the fastest known enzyme with a turnover number of 6 million.  It is especially abundant in plant storage organs and in liver.  It is effective over the pH range of 4.0 to 9.0 with the optimum pH of 7.6.  

2H2O2(aq)    catalase    2H2O(l)  + O2(g)

As an enzyme, its rate is affected by various factors, including pH.  Changes in pH alter the ionic charge of acidic and basic groups of the enzyme.  This therefore distorts the shape of the enzyme, including its active site, where substrates temporarily bind.  The efficiency of forming enzyme-substrate complexes is consequently lowered and the rate of activity decreases.  However, the enzyme can regain its most proficient shape, if it is not exposed to too extreme pHs, to return to its maximum rate of activity when placed back at the optimum pH.  

        

Plan:

The volume of oxygen produced during the catalysed break down of hydrogen peroxide can be measured in a graduated gas syringe whilst catalase, using potato, works at various pHs over a certain time.  The hydrogen peroxide should be added to the potato suspension in a flask to start the experiment.  Readings of the volume of oxygen produced is to be taken at set intervals during the experiment.  The pH of the catalase can be kept constant by a pH buffer solution of the required pH.  The average rate of decomposition can be calculated and a graph plotted of pH against rate.  This can then be analysed for the effect of pH on catalase to prove or disprove the hypothesis.

Alternative:  Use yeast, broad beans or liver, instead of potato.

Diagram:

Brief Method:

  1. Set up apparatus as shown in the diagram.
  2. Mince and weigh out potato to form a suspension with distilled water.
  3. Add buffer solution and warm mixture in water bath to optimum temperature of catalase.
  4. Check pH is still correct at the optimum temperature.
  5. Inject hydrogen peroxide onto potato and start timer.
  6. Record volume of oxygen produced every 10 seconds for the first second, and then every minute thereafter till 4 minutes has passed.
  7. Repeat with different pHs.

Apparatus:

        1 100 cm3 gas syringe (with rubber tubing)

        1 boiling tube

        1 250 cm3 beaker

        1 100 cm3 beaker

        1 10 cm3 graduated pipette (with filler)

        1 watch glass

        1 spatula

        2 thermometer

        1 2 cm3 plastic syringe

        1 10 cm3 measuring cylinder

        1 stop clock

        1 bung (with 2 holes for plastic syringe and to connect to gas syringe)

        1 clamp stand, clamp & boss

        1 electronic balance

        1 Bunsen burner

        1 tripod

        1 gauze

        matches

        narrow range indicator paper for pHs 4.5 – 8.5

        grater

        potato

        15 vol. hydrogen peroxide solution

        pH buffer solutions (pH 4.5, 5.5, 6.5, 7.0, 7.5, 8.5)

        distilled water

Variables:

        There are 4 main factors, which affect the activity of enzymes that need to be controlled.

  1. Temperature:  The reaction is to take place at the optimum temperature of catalase, in a 40oC water bath.
  2. Substrate concentration:  A fixed volume of one concentration of hydrogen peroxide is to be used each time the experiment is repeated.
  3. Enzyme concentration:  The concentration of catalase will be unknown but by using a fixed mass of potato, the concentration of the enzyme can be more constant.
  4. pH:  Buffer solutions of each pH to be tested keep the reaction system constant.
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Risk assessment:

Hydrogen peroxide: 

It is an oxidizer, which is not flammable but may start a fire if put in contact with organic materials, so keep the area well ventilated.  The maximum concentration that is safe to use in the laboratory is 20 vol.  Make sure eye protection and protective clothing is worn, as hydrogen peroxide is corrosive.  In case of any solution getting in contact with the eyes, flush it out with copious amounts of water and seek medical assistance.  Any contact with the skin should be avoided but in case there is any contact, wash ...

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This is a very good report on a practical that demonstrates a good understanding of scientific processes. 1. The background knowledge is good but needs referencing. 2. The preliminary test is also good. However, the conclusion from it needs to be readdressed. 3. The language used is appropriate throughout 4. The conclusion section has become confused about the information that should be included. 5. The evaluation is thoughtful and detailed. **** (4 stars)