GENERAL PROTEASE ASSAY-METHOD COUPLING SOLID-PHASE SUBSTRATE EXTRACTION AND CAPILLARY-ELECTROPHORESIS

Capillary electrophoresis with laser-induced fluorescence detection wa s used to develop a universal, highly specific protease assay. In this method, a peptide, biotinylated at the N-terminus, is labeled with fl uorescein at a lysine residue near the C-terminus. Impurities are remo ved from the fluorescence labeling mixture by solid-phase extraction o f the substrate on immobilized streptavidin, followed by extensive was hing. The purified fluorescent substrate is dissociated from the strep tavidin and incubated with the protease. The peptide sequence between the biotin and fluorescent label contains the cleavage sequence of the protease of interest. After cleavage, the fluorescent product does no t contain a biotin group. A second solid-phase extraction is used to r emove unreacted substrate to dramatically lower the background signal. The product is detected by capillary electrophoresis, which provides powerful discrimination against products Generated by nonspecific prot eases. With chymotrypsin as a test protease, product was detected with as little as 10 pg/mL(4.6 x 10(-13) M) chymotrypsin, or 5 amol of enz yme in the 10-mu L sample volume.