Abstract
A sensitive enzyme assay is described using substrates derivatized with metal-binding ligands. Enzymes catalyzed changes in the abilities of substrates to bind to the nonelectrochemiluminescent complex ruthenium(II) bis(bipyridyl), Ru(bpy)22+, to form electrochemiluminescent mixed-ligand complexes. A highly electrochemiluminescent complex was formed between Ru(bpy)22+and picolinic acid (1) but not picolinic acid ethyl ester (4), permitting the detection of4hydrolysis by pig liver esterase (PLE). Electrochemiluminescence (ECL) differences between Ru(bpy)22+mixtures of1and4were detected to a lower concentration limit of 65 pM. Under the conditions used in actual enzyme assays, it was possible to detect 4.4 pMPLE and the hydrolysis of 1.3 μM4. In a second assay, leucine aminopeptidase (LAP) hydrolyzed 8-(L)-leucylaminoquinoline (9) to leucine and aminoquinoline (10). A mixed-ligand complex formed between (9) and Ru(bpy)22+was substantially more electrochemiluminescent than a complex of Ru(bpy)22+and10. ECL differences between Ru(bpy)22+mixtures of9and10were detectable to 65 nM. Under actual enzyme assay conditions, 375 pMLAP could be detected as well as the hydrolysis of 1.3 μM9.
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