Lactoferrin-Mediated Formation of Oxygen Radicals by NADPH-Cytochrome P-450 Reductase System

Abstract

Superoxide generation in the NADPH oxidase reaction of NADPH-cytochrome P-450 reductase, demonstrated using the ESR spin trap, 5, 5-dimethyl-l-pyrroline-l-oxide, increased on the addition of lactoferrin. The NADPH-lactoferrin reductase activity was assessed in terms of NADPH oxidation and oxygen consumption. From Lineweaver-Burk plots, the Ka, and V_mBX for lactoferrin were estimated to be 13 /iM and 0.5 s¹, respectively. The liberation of iron from lactoferrin was proven with the use of bathophenanthroline and by the demonstration of bleomycin-dependent DNA degradation; lactoferrin was reduced by the enzyme in the presence of NADPH. During the reaction, the ESR spectrum of the spin trap adduct changed from one characteristic of DMPO-OOH to that of DMPO-OH. The conversion was ascribed to the reaction of hydrogen peroxide with reduced lactoferrin.