Karakawa Tomohiro, Sato Keizo, Muramoto Yosuke, Mitani Yoshihiro, Kitamado Masataka, Iwanaga Tatsuya, Nabeshima Tetsuji, Maruyama Kumiko, Nakagawa Kazuko, Ishida Kazuhiko, Sasamoto Kazumi
Division of Pharmacology and Therapeutics, Faculty of Medical and Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-Honmachi, Kumamoto 862-0973, Japan.
Biochem Biophys Res Commun. 2008 May 23;370(1):93-7. doi: 10.1016/j.bbrc.2008.03.048. Epub 2008 Mar 18.
Electron spin resonance using spin-trapping is a useful technique for detecting direct reactive oxygen species, such as superoxide (O2.-). However, the widely used spin trap 2,2-dimethyl-3,4-dihydro-2H-pyrrole N-oxide (DMPO) has several fundamental limitations in terms of half-life and stability. Recently, the new spin trap 2-diphenylphosphinoyl-2-methyl-3,4-dihydro-2H-pyrrole N-oxide (DPhPMPO) was developed by us. We evaluated the biological applicability of DPhPMPO to analyze O2.- in both cell-free and cellular systems. DPhPMPO had a larger rate constant for O2.- and formed more stable spin adducts for O2.- than DMPO in the xanthine/xanthine oxidase (X/XO) system. In the phorbol myristate acetate-activated neutrophil system, the detection potential of DPhPMPO for O2.- was significantly higher than that of DMPO (k(DMPO)=13.95M(-1)s(-1), k(DPhPMPO)=42.4M(-1)s(-1)). These results indicated that DPhPMPO is a potentially good candidate for trapping O2.- in a biological system.
利用自旋捕获的电子自旋共振是检测直接活性氧物种（如超氧阴离子（O2.-））的有用技术。然而，广泛使用的自旋捕获剂2,2-二甲基-3,4-二氢-2H-吡咯N-氧化物（DMPO）在半衰期和稳定性方面存在一些基本限制。最近，我们开发了新型自旋捕获剂2-二苯基膦酰基-2-甲基-3,4-二氢-2H-吡咯N-氧化物（DPhPMPO）。我们评估了DPhPMPO在无细胞和细胞系统中分析O2.-的生物学适用性。在黄嘌呤/黄嘌呤氧化酶（X/XO）系统中，DPhPMPO对O2.-的反应速率常数比DMPO大，并且形成的O2.-自旋加合物更稳定。在佛波酯激活的中性粒细胞系统中，DPhPMPO对O2.-的检测潜力明显高于DMPO（k（DMPO）=13.95M(-1)s(-1)，k（DPhPMPO）=42.4M(-1)s(-1)）。这些结果表明，DPhPMPO是在生物系统中捕获O2.-的潜在良好候选物。
