Analysis of prostaglandin G/H synthase-2 inhibition using peroxidase-induced luminol luminescence.

The inducible form of the heme-protein prostaglandin G/H synthase (PGHS-2 or COX-2) has been established as a pivotal enzyme in the cascade of events leading to inflammation, hyperalgesia, and pyresis and represents a major therapeutic target in inflammatory disease. Accordingly, we have exploited the heme-catalyzed hydroperoxidase activity of recombinant hCOX-2 to generate luminescence in the presence of luminol, or a cyclic naphthalene hydrazide, and the substrate arachidonic acid. Arachidonate-induced luminescence was shown to be an index of real-time catalytic activity and demonstrated the turnover inactivation of the enzyme. Luminol luminescence was proportional to hCOX-2 concentration and gave accurate Km determinations for arachidonate. Inhibition of hCOX-2 activity, measured by luminescence, by a variety of selective (for COX-2) and nonselective inhibitors showed rank orders of potency similar to those observed with other in vitro and whole cell methods using the recombinant protein. The sensitivity of the luminescence assay also allowed determination of inhibitor potency at substrate concentrations below Km, distinguishing competitive inhibitors such as ibuprofen from time-dependent inhibitors such as DuP-697. Finally the use of higher quantum-yielding luminol analogues allowed measurement of cyclooxygenase activity at extremely low substrate and protein concentrations, enabling a variety of novel assay formats.