Protease
assay
In certain fruits, such as pineapples
and mangoes, the flesh contains protein-digesting enzymes (proteases). These
may play a part in helping to soften the fruit tissues as the fruit ripens,
making it even more attractive to animals that might disperse the seeds. So
perhaps the activities of these proteases enzymes will increase during the ripening
process.
Applications of plant proteases,
such as ‘bromelain’ from the stems and fruits of pineapples, include
uses in the pharmaceutical industry as a blood anti-coagulant, and in the prevention
of proteinaceous hazes in chill-proof beers!
The
assay
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Protease enzymes are
added to a milky colloidal suspension of egg albumen. As the protease
enzymes digest the suspended particles of proteins, the mixture becomes
more transparent. The absorbance changes in the reaction mixture are followed
with a colorimeter. The protease content, for example in extracts of fruits,
can be assayed by measuring the rate at which the solution of egg albumen
and extract becomes clearer.
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Preparation of
the egg albumen colloidal suspension (enzyme substrate)
- Separate the white of
a single egg into a 250 cm3 beaker and add 150 cm3
tap water, stirring the mixture thoroughly. The mixture becomes quite cloudy
as a result of the denaturation of some of the egg albumen by the water.
- Place the beaker on a
tripod and gauze and heat with a bunsen until the mixture boils, stirring
it regularly.
- Allow the mixture to
cool, then decant it through two or three layers of muslin into another beaker.
This creates a homogenous milky colloidal solution.
Carrying
out the reaction
- Select a test-tube that fits
into the colorimeter. Add 2 cm3 of an appropriate buffer (e.g.
pH7), followed by 2 cm3 of the albumen substrate solution
and 1 cm3 of fruit extract.
- Mix the contents of the colorimeter
tube, and place it in the colorimeter (previously zeroed using a tube with
4 cm3 buffer and 1 cm3 of fruit extract).
Read the absorbance and note the time.
- Place the reaction mixture(s)
in a water bath at 30°C to promote the activity of any protease enzymes
present.
- Take further absorbance readings
at regular time intervals (say every 5 or 10 minutes), until no further
change (decrease) in absorbance is detected.
- Plot a graph of the change
(decline) in absorbance against time. Measuring the time taken for a 50% reduction
in absorbance value gives an indication of the protease activity of the original
fruit extract.
Ideas for investigations
with this system (with fruits and vegetables)
- Follow the changes in protease
enzymes during ripening
- Compare the effects of pH on
proteases from different fruits
- Compare the effects of temperature
on proteases from different fruits
- Investigate the presence of
protease-inhibitors in the seeds of legumes
- Monitor the release of amino
acids from the digested proteins, using paper chromatography
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