Patent ID: 7727739

Claim:
In a method of measuring the activity of an enzyme in cleaving a substrate, said substrate including at least one fluorescent label bound to a polymer which includes one or more tyrosine, tryptophan, phenoxy, indolyl or nitro-phenylalanine moieties, said moieties being separated from said at least one fluorescent label by a linkage group cleavable by said enzyme, the improvement comprising the steps of: i) measuring the fluorescence lifetime of the at least one label of the substrate in a reaction buffer which is suitable for said enzyme to cleave said substrate; ii) adding said enzyme to said reaction buffer; and iii) measuring any increase in fluorescence lifetime of the at least one fluorescent label following step ii); wherein said increase in fluorescence lifetime indicates substrate cleavage and is used to determine enzyme activity; further wherein said at least one fluorescent label is an acridone dye of formula: wherein: groups R 2 and R 3 are attached to the Z 1 ring structure and groups R 4 and R 5 are attached to the Z 2 ring structure; Z 1 and Z 2 independently represent the atoms necessary to complete one or two fused ring aromatic or heteroaromatic systems, each ring having five or six atoms selected from carbon atoms and optionally no more than two atoms selected from oxygen, nitrogen and sulphur; R 1 , R 2 , R 3 , R 4 and R 5 are independently selected from hydrogen, halogen, amide, hydroxyl, cyano, amino, mono- or di-C 1 -C 4 alkyl-substituted amino, sulphydryl, carbonyl, C 1 -C 6 alkoxy, aryl, heteroaryl, C 1 -C 20 alkyl, aralkyl; the group -E-F where E is a spacer group having a chain from 1-60 atoms selected from the group consisting of carbon, nitrogen, oxygen, sulphur and phosphorus atoms and F is a target bonding group; and the group —(CH 2 —) n Y where Y is selected from sulphonate, sulphate, phosphonate, phosphate, quaternary ammonium and carboxyl and n is zero or an integer from 1 to 6 further comprising the use of a plurality of different substrates each bound to a plurality of different labels, wherein each said label is individually distinguishable from the others by its unique fluorescence emission and/or its fluorescence lifetime, thereby enabling simultaneous measurement of a plurality of enzyme cleaving activities.