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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
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. |
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.
- 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.
- 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 pr
- Follow the changes in protease enzymes during ripening
- 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.
