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enzymes explained

- Action of pepsin on proteins

The substrate used in this experiment consists of white of egg (albumen). It has been heated in order to solidify it, thus denaturing it - permanently damaging its protein structure - and it has also been finely divided and suspended in water to form an opaque white liquid.
The enzyme pepsin is from the mammalian digestive system. Depending on the conditions in the surrounding medium, it may break down (digest) the egg white to different extents, thus clearing the liquid.
During this practical session you will be seeing the effect of altering the pH on the proteolytic activity of the enzyme.

Preparation of a series of different pHs

CARE! Wear eye protection when handling acids. Do not use pipettes for any other purpose.

1) Place 6 test-tubes in a rack, and label them 1-6.

2) Using a measuring cylinder, put 10 ml of egg white suspension into tubes 1-6.

3) USING ONE PIPETTE, add 2 ml acid to tube 1 THEN ..

4) Add 1 ml acid to tube 2 THEN ..

5) Add 0.5 ml acid to tube 3 THEN ..

6) Add 2 ml acid to tube 5 THEN PLACE PIPETTE IN WASH.

7) Transfer the tubes to a water bath at 37 °C.

8) Leaving the tubes in place in the waterbath, add 3 ml pepsin to tubes 1-4 only. Start timing.

9) Insert a FRESH PIPETTE into each of tubes 1-6 and mix the contents. Take the pH of each tube using universal indicator paper (or narrow range version), and record the result in the table.

10) Leave the tubes in the waterbath "for some time". Check for clearing at regular intervals. Record this below - under "time results first seen". In the meanwhile, you have the opportunity to carry out the test procedure overleaf which will tell you more about these substances.

Tube number 1 2 3 4 5 6
Contents quantity /ml
TREATMENTS egg white suspension 10 10 10 10 10 10
acid 2 1 0.5 - 2 -
pepsin 3 3 3 3 - -
RESULTS measured pH value ............ ............ ............ ............ ............ ............
time results first seen ............ ............ ............ ............ ............ ............
result at end of expt ............ ............ ............ ............ ............ ............

click for photographs

What is meant by the term proteolytic ?

> breaking down proteins into simpler substances

What sort of enzyme is pepsin, i.e. what category does it belong to?

> protease

What sort of substance will be produced as a result of its action on egg white?

> amino acids

What do you think that substance would do in water, and what would it look like?

> dissolve to give a clear solution

Whereabouts in the body is pepsin produced?

> stomach

What sort of physical conditions exist there?

> acidic ( also warm, wet)

What do you think is the role of hydrochloric acid in this experiment?

> to alter pH

Which tube or tubes show a change first?


What combination do the tubes that give a positive result include?

> pepsin + (lots of) acid

Carefully compare the results from these tubes with the others. Write down your conclusion from this experiment. This should include answers to questions such as :

What does pepsin do to the protein in egg white? What other factors are needed ? Are they absolutely necessary?

> protein is broken down (made into soluble amino acids) by a combination of the enzyme pepsin and acid, not either works alone


Whilst you are waiting for results from the above procedure, you should perform the biuret test (separately) on the spare pepsin, egg white and (1%) amylase you have been using:

What is the biuret test used to identify?

? > protein

Pour about 10 ml of sodium (or potassium) hydroxide into a boiling tube.

CARE! Wear eye protection when handling alkalies.

Add about 1 ml of copper sulphate solution; the colour should change to a deeper blue. Mix carefully. This is biuret reagent.

Place about 2.5 ml of each substance to be tested into a tube in the rack.

To each add about 2.5 ml of the blue mixture (biuret reagent) produced above.

Wait for a different colour to develop, compared with the leftover untouched mixture.

Test substance Resulting colour Conclusion : substances present
pepsin purple/lilac protein
egg white (greyish) mauve protein
amylase purple/lilac protein

click for photographs What is your general conclusion from these tests?

>The enzymes pepsin and amylase are/contain protein - also egg white is a protein

Finally: Pour away the contents of ALL the tubes down the sink, then rinse the tubes with a gentle flow of cold water before placing them in the washing up bowl.

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