Time-resolved europium fluorescence in enzyme activity measurements: a sensitive protease assay

A method for incorporating into proteins a nonradioactive Eu3+ label, which exhibits fluorescence of a long decay time in the presence of suitable ligands, is described. As an example of the use of this label the method has been developed to work as a sensitive protease assay. By hydrolyzing the Eu3+-labeled casein, bound to an insoluble matrix (Sepharose 4B or Affi-Gel 10), with proteases and measuring the Eu3+ released with a pulsed time-resolved fluorometer it was possible to detect as low as 2.5, 1.0, or 1.0 ng of alpha-chymotrypsin, trypsin, or subtilisin, respectively.