Enzymatic analysis using substrates that yield fluorescent precipitates

A method for detecting the activity of an enzyme, comprising:

• a) combining a sample of cells suspected of containing the enzyme, with a substrate of the formula:
• (BLOCK-O)--X.sub.fl
• where BLOCK is a blocking group that is a monovalent moiety derived by removal of a hydroxy group from phosphate, from sulfate or a biologically compatible salt thereof;
• or a monovalent moiety derived by removal of a hydroxy group from an alcohol or from a carboxy group of an aliphatic, aromatic or amino acid or of a peptide;
• or a monovalent moiety derived by removal of an anomeric hydroxy group from a mono- or polysaccharide;
• and is capable of being cleaved from the remainder of the substrate by action of the specific enzyme resulting in a fluorescent precipitate of the formula:
• X--O--X.sub.fl
• where X.sub.fl has the structure:
• ##STR26## where Z is --(C.dbd.O)-- or --CH.dbd.(methine) and n=1 or 0;
• where W is (CH.sub.3).sub.2 C, --CH.sub.2 --, --CH.dbd., S, O, or --(N--R)--, wherein R is H or lower alkyl containing 1-4 carbons;
• and
• where each aromatic ring is optionally modified by substitution of one or more hydrogens on an aromatic carbon by substituents that are halogen, nitro, lower alkyl (1-4 carbons), or alkoxy (1-4 carbons), or any combination thereof;
• in aqueous buffer at a concentration of substrate between about 0.1 mM and about 5 mM at a pH less than about 8.5 for greater than 5 minutes;
• b) exposing the precipitate to a light source with a wavelength greater than about 300 nm;
• and
• c) detecting fluorescence of the precipitate at greater than about 400 nm.

A method for detecting the activity of an enzyme conjugate, comprising:

• a) combining a sample containing the enzyme that is incorporated in a specific binding complex, with a substrate of the formula:
• (BLOCK-O)--X.sub.fl
• where BLOCK is a blocking group that is a monovalent moiety derived by removal of a hydroxy group from phosphate, from sulfate or a biologically compatible salt thereof;
• or a monovalent moiety derived by removal of a hydroxy group from an alcohol or from a carboxy group of an aliphatic, aromatic or amino acid or of a peptide;
• or a monovalent moiety derived by removal of an anomeric hydroxy group from a mono- or polysaccharide;
• and is capable of being cleaved from the remainder of the substrate by action of the specific enzyme resulting in a fluorescent precipitate of the formula:
• H--O--X.sub.fl
• where X.sub.fl has the structure:
• ##STR27## where Z is --(C.dbd.O)-- or --CH.dbd.(methine) and n=1 or 0;
• where W is (CH.sub.3).sub.2 C, --CH.sub.2 --, --CH.dbd., S, O, or --(N--R)--, wherein R is H or lower alkyl containing 1-4 carbons;
• and
• where each aromatic ring is optionally modified by substitution of one or more hydrogens on an aromatic carbon by substituents that are halogen, nitro, lower alkyl (1-4 carbons), or alkoxy (1-4 carbons), or any combination thereof;
• in aqueous buffer at a concentration of substrate between about 0.1 mM and about 5 mM at a pH less than about 8.5;
• b) exposing the precipitate to a light source with a wavelength greater than about 300 nm;
• and
• c) detecting fluorescence of the precipitate at greater than about 400 nm.