The slow breakdown of albumen proteins gives good results with continuous colorimetry and is well suited for the study of the effect of pH on enzyme activity.

Pepsin (EC is a proteolyic (protein splitting) enzyme produced in the gastric glands lining the stomach of vertebrates. It is produced as pepsinogen, a larger molecule that is activated by hydrochloric acid to the smaller pepsin molecule.

Pepsin can only work in a low pH environment, (optimum pH~1.8)

Pepsin does not break the bonds between every amino acid so the products of the reaction are polypeptides of varying lengths.

Pepsin can conveniently be assayed using a cloudy suspension of egg white (albumen), which will slowly clear as the albumen protein is broken down into small, soluble polypeptides. The preparation of a suitable suspension is critical for the success of this assay (see Methods).


mask Powdered enzymes may provoke allergic reactions. Solutions should be prepared in a fume cupboard, or with care while wearing a dust mask. Skin contact with powdered enzymes and prepared solutions should be avoided. gloves

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Absorbance vs Time

    2.8cm3 egg white suspension adjusted to pH2.1 at 25°C + 0.2cm3 of 5% pepsin solution.
    The rate of the reaction shown below is the slope of the line in the region between absorbance 0.5 and 0.2, given as -0.003575 absorbance units per second.

    absorbance vs time

Effect of pH on reaction rate

    2.8cm3 egg white suspension at 25°C + 0.2cm3 of 5% pepsin solution.

    pepsin pH

Effect of temperature on reaction rate

    2.8cm3 egg white suspension adjusted to pH 1.8 + 0.2cm3 of 5% pepsin solution.
    Warm both solutions to the reaction temperature before mixing the egg white suspension with the enzyme.
    The pH of the egg white suspension should be adjusted to pH 1.8 after it has reached the required temperature.

    pepsin temperature

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To prepare an suspension of egg white

    eye-protection WEAR EYE PROTECTION

    • separate the white from the yolk – depending on the size of the egg this will probably be about 30cm3
    • add 9 volumes of water – 270cm3 to a 30cm3 egg (this seems to work well with a fresh egg, but older eggs may need less water to achieve a good, dense suspension.)
    • stir gently to disperse the egg white
    • stand the mixture in a bath of hot water, with gentle stirring, for 5-10 minutes, (do not boil this – the suspension will form at around 70°C)
    • pour the mixture through 2-4 layers of butter muslin cloth to remove large particles.

    You should be left with an opaque suspension. This can be diluted 1:1 with water or buffer for use

    This is a rather unpredictable outcome. Different eggs give different suspensions and if the suspension is not sufficiently opaque, with an absorbance greater than about 0.7, the results are likely to be disappointing. Reducing the ratio of water to egg white will probably help.

Adjusting the pH

    The optimum pH for pepsin is around 1.8

    The egg white suspension can be acidified as it is, or diluted 1:1 with water first.
    Adding 0.15cm3 of 2M HCl to 10cm3 of the diluted suspension will give about the right pH.

    To investigate the optimum pH a range of different solutions can be set up as indicated in the table. These are only valid at 25°C, the pH can vary significantly with changes in temperature.

    To 50cm3 of a 1:1 mixture of egg white suspension and water add 2M HCl according to the table below.

    pH (at 25°C) Volume of 2M HCl (cm3)
    1.1 3.0
    1.3 2.0
    1.5 1.2
    1.7 0.8
    1.9 0.5
    2.1 0.4
    2.3 0.3

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