A NITROXYL SYNTHASE CATALYTIC ANTIBODY

An antibody raised against acridinium hapten 1 is shown to catalyze th e retro Diels-Alder reaction of the anthracene-HNO cycloadduct 2 to re lease anthracene 4 and nitroxyl (HNO). Nitroxyl is oxidized to nitric oxide (NO) in the presence of superoxide dismutase. Since the enzyme s uperoxide dismutase is ubiquitous in vivo, this catalytic antibody sys tem may be equivalent to NO-synthase. Antibody catalysis is triggered by recognition of the phenyl rings in hapten 1 at an angle near that o f the transition state of the retro Diels-Alder reaction of 2. Acridin ium hapten 1 is in chemical equilibrium with its conjugate Lewis base 9-hydroxyacridane 1-OH (pK similar to 8.2). Catalytic antibody 9D9 (K- M(2) = 100 mu M, k(cat) = 0.07 min(-1), k(uncat) = 3 x 10(-4) min(-1)) is the result of an heterologous immunization and binds both 1 (K-i = 16.6 mu M at pH 6.1) and 1-OH (K-i = 0.9 mu M at pH 9.0). The more ti ghtly bound neutral conjugate base 1-OH probably represents a more acc urate mimic of the transition state of the retro Diels-Alder process.