Luminescence amplification by enzymatic Eu2+ oxidation to Eu3+ for time-resolved peroxidase activity measurement.

: A novel peroxidase activity assay was developed for horseradish peroxidase (HRP) and myeloperoxidase (MPO), in which substrate Eu(2+) was catalytically oxidized to Eu(3+), and the Eu(3+) luminescence was enhanced by the addition of sensitizer 4,4'-bis(1″,1″,1″,2″,2″,3″,3″-hepatafluoro-4″,6″-hexanedione-6″-yl)chlorosulfo-o-terphenyl (BHHCT) for time-resolved measurement of the BHHCT-Eu(3+) complex. Since BHHCT-Eu(3+) has a long lifetime (more than 500 μs), typical of Eu(3+) oxidation state, and the emission wavelength (615 nm) is totally different from those of Eu(2+) complexes, time-resolved luminescence measurement of the Eu(3+) complex enabled suppressed background and high signal/background ratio. The present method was successfully applied to monitor the oxidative stress level, which is closely associated with peroxidase activity level, in rat heart muscle homogenates. Notable parallel temporal change was observed for peroxidase activity and 4-hydroxynonenal (HNE) concentration after lipopolysaccharide (LPS) injection for induction of oxidative stress in rats. Such a relation does not contradict the oxidative stress mechanism that HNE is produced via lipid peroxidation, which is caused by the (•)OH radical generated by peroxidase activity.