Inactivation of lactoperoxidase by 4-chloroaniline

Chlorinated anilines are metabolites of a variety of herbicides, and these compounds are incorporated covalently into plant lignin. Recent studies have shown that chlorinated anilines in lignin become bioavailable in the digestive system of experimental animals. It is, therefore, of importance to assess the interaction of these compounds with potential target molecules. Bovine lactoperoxidase (LPX) oxidized 4-chloroaniline and was inactivated. Kinetic analysis revealed that inactivation was time dependent and exhibited saturation kinetics. The dissociation constant (K(I)) for LPX and 4-chloroaniline was 1.5 mM, the pseudo-first-order rate constant for inactivation (k(inact)) was 6.9 X 10(-2) s-1, and t1/2 for inactivation was 10 s. The partition coefficient was found to be approximately 115 regardless of which of the cosubstrate (4-chloroaniline or hydrogen peroxide) concentrations was varied. Extensive ( 40:1) incorporation of [14C]-4-chloroaniline into LPX during turnover was demonstrated, and inactivation followed first-order kinetics only during the first 3 min of inactivation. These results show that 4-chloroaniline is a metabolically activated inactivator of LPX rather than a mechanism-based inactivator. Although LPX is inactivated by 4-chloroaniline in vitro, kinetic constants associated with this inactivation demonstrate that, prior to inactivation, LPX has only moderate affinity for 4-chloroaniline. Furthermore, these analyses suggest that it is improbable that the concentrations of 4-chloroaniline sufficient to cause inactivation of LPX would occur in vivo as a result of ingesting residual amounts of 4-chloroaniline present in foliage