Analytical Sciences

Abstract − Analytical Sciences, 29(10), 971 (2013).

Luminescence Amplification by Enzymatic Eu2+ Oxidation to Eu3+ for Time-Resolved Peroxidase Activity Measurement
Kazuko MATSUMOTO,*1 Hiroko KIMURA,*2 Nobuko KON,*2 Ken-ichi YOSHIDA,*3 Mitsuru ABO,*4 and Etsuro YOSHIMURA*5
*1 Vision Development Co., Ltd., 2-8-21 Kyobashi, Chuo, Tokyo 104-0031, Japan
*2 Department of Forensic Medicine, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo, Tokyo 113-8421, Japan
*3 Department of Forensic Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033, Japan
*4 Department of Agricultural Chemistry, Faculty of Agriculture, Meiji University, Kawasaki, Kanagawa 214-8571, Japan
*5 Department of Applied Biological Chemistry, The University of Tokyo, Yayoi, Bunkyo, Tokyo 113-8657, Japan
A novel peroxidase activity assay was developed for horseradish peroxidase (HRP) and myeloperoxidase (MPO), in which substrate Eu2+ was catalytically oxidized to Eu3+, and the Eu3+ 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-Eu3+ complex. Since BHHCT-Eu3+ has a long lifetime (more than 500 μs), typical of Eu3+ oxidation state, and the emission wavelength (615 nm) is totally different from those of Eu2+ complexes, time-resolved luminescence measurement of the Eu3+ 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.