Specific binding reactions monitored with ligand-cofactor conjugates and bacterial luciferase

Abstract

A unique rapid method for assaying specific binding reactions with ligand-cofactor conjugates and a bioluminescent reaction is described. Biotin and 2,4-dinitrofluorobenzene were coupled covalently to the free amino residue of nicotinamide 6-(2-aminoethylamino) purine dinucleotide (AENAD) to produce the enzymatically active conjugates biotinyl-AENAD and DNP-AENAD. After reduction with alcohol dehydrogenase and ethanol these two conjugates were measured quantitatively by means of light produced in a bioluminescent reaction employing luciferase from Photobacterium fisheri. Light production by biotinyl-AENADH and DNP-AENADH was inhibited by the specific binding proteins avidin and antibody to DNP, respectively. The counter ligands, biotin and DNP-6-aminocaproate, reversed the inhibition by the respective binding proteins in competitive binding reactions. Thus, specific binding reactions can be assayed rapidly without separation of free and bound labeled ligands.