A Simple Method Involving Aqueous Two-Phase Separation-Systems for Activity Determination of Enzymes Hydrolyzing Macromolecular Substrates

A new general procedure for quantitation of enzymes degrading macromolecular substrates is described. With the use of chromogenic substrates combined with separation of substrate and product by aqueous two-phase systems, rapid separation and accurate measurements can be achieved. Furthermore, in competitive substrate assays using constant amounts of enzyme and chromogenic substrate in combination with a varying amount of native substrate, unknown concentrations of native substrate can easily be determined. The principle is illustrated here by a system consisting of the enzyme, dextranase; the chromogenic substrate, blue dextran; and the native substrate, dextran.