Azocase test

The azocasein test is a test method for determining the enzyme activity of peptidases .

Basics

Casein is the most important protein in milk in terms of quantity and makes up 80% of the total protein content. An azo dye is an organic pigment that contains one or more azo groups (-N = N-) as a coloring group that combine two aromatics (Ar) to form compounds of the Ar-N = N-Ar type.

Azo compounds absorb light in the visible spectrum and therefore appear colored. The absorption wavelength depends on the nature of the substituents around the azo group. Azocasein is yellowish in color.

principle

A casein linked to azo dye serves as a proteinase substrate. The determination principle is based on the conversion of the substrates by proteinases, which release dye-labeled peptides which can no longer be precipitated or filtered.

In the absence of a proteinase, the azocasein remains intact. It precipitates as an acid-insoluble macromolecule by adding trichloroacetic acid and can be centrifuged off as a yellow precipitate, whereby the supernatant remains colorless.

In the presence of a protease, on the other hand, the azocasein is cut into small peptides which are acid-soluble and therefore cannot be centrifuged off and give the supernatant a yellowish color.

The determined absorbance is proportional to the proteinase activity.