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Lucigenin-Derived Chemiluminescence in Intact Isolated Mitochondria

A. G. Kruglov*, I. S. Yurkov, V. V. Teplova, and Yu. V. Evtodienko

Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Institutskaya ul. 3, Pushchino, Moscow Region, 142290 Russia; fax: (0967) 79-0553; E-mail: kruglovag@mail.ru

* To whom correspondence should be addressed.

Received October 24, 2001; Revision received November 28, 2001
There are many data both in favor and against the use of lucigenin as a probe for superoxide anion (SA) in mitochondria, cells, and simple enzymatic systems. In the present work high concentrations (50-400 µM) of lucigenin were used for continuous recording of rapid and reversible changes in the SA level in intact isolated mitochondria. The SA level in the presence of lucigenin rapidly and reversibly changed during the transition of the mitochondria from one functional state to another: under conditions of ATP synthesis from ADP and Pi, of Ca2+ accumulation, and of reverse electron transfer. Induction of a Ca2+,cyclosporin A-sensitive pore in mitochondria completely suppressed the lucigenin-derived chemiluminescence (LDC). The electron transfer in the Q-cycle of the respiratory chain complex III and high electric potential difference across the inner membrane of mitochondria were obligatory conditions for generation of a SA-dependent chemiluminescent signal. Based on our own and literature data, a scheme of LDC generation is suggested. The origin of superoxide anion detected in intact mitochondria with lucigenin is discussed.
KEY WORDS: lucigenin, superoxide anion, mitochondria, Ca2+, mPTP, reverse electron transfer, complex III