Specific binding reactions were monitored by a new enzymatic method which employs cofactor labeled ligands. The ligands biotin and 2+dinitrofluorobenzene were coupled covalently to a derivative of NAD, nicotinamide 6-(2-aminoethylamino) purine dinucleotide (AENAD) to provide cofactor conjugates which were active with several dehydrogenases. The conjugates were cycled in reactions employing lactic dehydrogenase and diaphorase and cycling rates were determined by spectrophotometric measurement of a reaction product, reduced thiazolyl blue. The cycling of biotinyl-AENAD was inhibited specifically by avidin, a biotin-binding protein. This inhibition was reversed by biotin in competitive binding reactions. Similarly, the cycling rate of 2,4dinitrophenyl-AENAD was inhibited by antibody to the 2,4-dinitrophenyl residue and 2,4-dinitrophenyl-6-aminocaproate reversed this inhibition. Thus, the activity of a cofactor coupled to a ligand is modified by a binding protein specific for the ligand. This phenomenon provides a versatile method for conducting specific binding assays without separation of free and bound labeled ligand.
Antibodies and a number of other proteins bind hormones, metabolites, and various other compounds with high affinity and specificity. The specificity of such proteins forms the basis for highly sensitive competitive binding assays. Generally, radiolabeled ligands are used to monitor binding reactions by methods which require separation of free and protein bound labeled ligands.
Recently, the need for such separation was circumvented through the use of enzyme-ligand conjugates (1). A ligand was coupled covalently to an enzyme at a position near the active site. The enzymic activity of this conjugate was diminished when the ligand was bound to an antibody. In competitive binding assays, levels of unconjugated ligand were related to enzymic activity.
We have developed a new, convenient, and highly versatile monitoring technique which employs cofactor labeled ligands and takes advantage