Continuous Measurement of the Lipoxygenase-Catalyzed Oxidation of Unsaturated Lipids Using the Monomolecular Film Technique

Abstract

This paper presents the first detailed kinetic investigation involving the continuous measurement of the soybean lipoxygenase 1 (LOX1)-catalyzed oxidation of unsaturated lipids using the monomolecular film technique at an argon/water interface. The presence of oxidation products in the monolayer is qualitatively detected, at a constant area, by an increase in the monolayer surface pressure. Alternatively, the rate of lipid oxidation can be measured, at a constant surface pressure, by a backward movement of the mobile barrier, due to the oxidation-dependent increase in the monolayer area. For instance, the LOX1-catalyzed oxidation of 1,2-di[cis-9,12-octadecadienoyl]-sn-glycero-3-phosphocholine (diC18:2PC) monolayer was found to be characterized by a time dependent increase in the monolayer area, at constant surface pressure. However, the increase in the monolayer area was thought to be caused first by the penetration of the enzyme into the interface, and secondly, by the formation of hydroperoxides at the interface, due to the LOX1-catalyzed oxidation of the diC18:2PC film. The rate of the LOX1-catalyzed oxidation of diC18:2PC film was measured by subtracting the increase in the area due to the LOX1-penetration into the non-oxidizable 1,2-di[cis-9-octadecenoyl]-sn-glycero-3-phosphocholine (diC18:1PC) film from the increase in the area due to LOX penetration and oxidation of the diC18:2PC film. At a constant optimum surface pressure of 1 mN m(-1), similar initial rates of LOX1-catalyzed oxidation are observed with both linoleic acid methyl ester (C18:2) and diC18:2PC. It is worth noting that the surface density of C18:2 acyl chains is also similar in both films. We observed that a phosphatidylcholine (PC) film with two potentially oxidizable chains (e.g., diC18:2PC) is oxidized at a rate which is twice that obtained with a PC containing a single oxidizable chain (e.g., 1-hexadecanoyl-2-[cis-9,12-octadecadienoyl]-sn-glycero-3-phosphocholine). The enzymatic lipid oxidation seems to occur when the monolayer is in the expanded state. This expanded state may possibly result in vivo from the lipolysis of a biomembrane and consequently lipolysis and lipid oxidation are coupled at the membrane level.