A melatonin-based fluorescence method for the measurement of mitochondrial complex III function in intact cells

Authors

  • Jian-Liang Fu,

    1. Geriatric Research, Education and Clinical Center, Audie L Murphy Division, South Texas Veterans Health Care System, San Antonio, TX, USA
    2. Department of Neurology, Shanghai Jiaotong University Affiliated Sixth People's Hospital, Shanghai, China
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    • These authors contributed equally to this work.
  • Hong-Mei Zhang,

    1. Geriatric Research, Education and Clinical Center, Audie L Murphy Division, South Texas Veterans Health Care System, San Antonio, TX, USA
    2. Department of Clinical Oncology, Xijing Hospital, The Fourth Military Medical University, Xi'an, China
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    • These authors contributed equally to this work.
  • Hua Zhang,

    1. Geriatric Research, Education and Clinical Center, Audie L Murphy Division, South Texas Veterans Health Care System, San Antonio, TX, USA
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  • Amrita Kamat,

    1. Geriatric Research, Education and Clinical Center, Audie L Murphy Division, South Texas Veterans Health Care System, San Antonio, TX, USA
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  • Chih-Ko Yeh,

    1. Geriatric Research, Education and Clinical Center, Audie L Murphy Division, South Texas Veterans Health Care System, San Antonio, TX, USA
    2. Department of Comprehensive Dentistry, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA
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  • Bin-Xian Zhang

    Corresponding author
    1. Department of Comprehensive Dentistry, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA
    • Geriatric Research, Education and Clinical Center, Audie L Murphy Division, South Texas Veterans Health Care System, San Antonio, TX, USA
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Address reprint requests to Bin-Xian Zhang, Geriatric Research, Education and Clinical Center, South Texas Veterans Health Care System, Audie L Murphy Division, 7400 Merton Minter Blvd, San Antonio, TX 78229, USA.

E-mail: zhangb2@uthscsa.edu

Abstract

Mitochondrial complex III (MC-3) plays a pivotal role in electron transfer and oxidative phosphorylation. Impaired MC-3 functions may contribute to a variety of diseases by interrupting normal bioenergetics and increasing reactive oxygen production and oxidative stress. Currently, MC-3 function is assessed by measuring the cytochrome c reductase activity spectrophotometrically in isolated mitochondria or MC-3. The cytoplasmic microenvironment critical for mitochondrial complex functions may be depleted during these isolation processes. The development of a reliable method to measure MC-3 activities in intact cells or tissues is highly desirable. This report describes a novel fluorescence-based method to assess MC-3 functions, i.e., Qi site electron transfer, in the intact cells. Human mesangial and teratocarcinoma NT2 cells were used to demonstrate that melatonin-induced oxidation of 2′,7′-dichlorodihydrofluorescein (H2DCF) was inhibited by antimycin A, the MC-3 Qi site-specific inhibitor, but not by myxothiazol, the MC-3 Qo site-specific inhibitor, nor rotenone, the mitochondrial complex I inhibitor. These results indicate that melatonin-induced oxidation of H2DCF is reflecting MC-3 Qi site electron transfer activities. Modifying structures of the side groups at the R3 and R5 positions of the indole ring of melatonin diminished its efficacy for inducing H2DCF oxidation, suggesting a specific interaction of melatonin with the MC-3 Qi site. These results suggest that the fluorogenic property of melatonin-induced H2DCF oxidation provides a MC-3 Qi site electron transfer-specific measurement in intact cells. Interestingly, using this method, the Qi site electron transfer activity in transformed or immortalized cells was found to be significantly higher than the nontransformed cells.

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