Comparison of recombinant cyclooxygenase‐2 to native isoforms: aspirin labeling of the active site

Abstract

The search for isoform‐specific enzyme inhibitors has been the focus of much recent research effort. Towards this goal, human recombinant cyclooxygenase‐2 (EC 1.14.99.1, prostaglandin H synthase) was expressed in insect cells and purified to > 98% purity. Recombinant enzyme was characterized both by physical methods and activity measurements and shown to be fully active with kinetic properties similar to native COX‐2 and COX‐1. After detergent extraction, the enzyme had hydrodynamic properties indistinguishable from native bovine COX‐1 and corresponded to the enzyme dimer as measured with size‐exclusion chromatography. Peptide mapping via Lys‐C protease identified a site of N‐linked glycosylation and the aspirin covalent modification site. In the presence of heme, aspirin‐specifically acetylated Ser‐516. The enzyme will be suitable for biophysical studies and may lead to isoform‐specific enzyme inhibitors.