MiP2005: Session 7

Mitochondrial Physiology Network 10.9: 92 (2005) - download pdf

 

MitoSOD: a novel mitochondria-targeted superoxide dismutase mimetic.

Meredith F Ross, GF Kelso, RAJ Smith, MP Murphy

MRC Dunn Human Nutrition Unit, Cambridge CB2 2XY, UK; Department of Chemistry, University of Otago, Dunedin, New Zealand. - mfr@mrc-dunn.cam.ac.uk

    Superoxide (O2•-) is the proximal reactive oxygen species (ROS) generated by the mitochondrial respiratory chain, which is the major source of ROS in the cell. It is estimated that up to 1% of respiratory chain electrons generate superoxide (the one-electron reduction product of molecular oxygen) instead of contributing to the reduction of oxygen to water. Once generated, mitochondrial superoxide can react to form various other ROS, including hydrogen peroxide, peroxynitrite and the hydroxyl radical; together, these ROS can cause oxidative damage to all classes of macromolecules. For this reason, mitochondrial ROS are strongly implicated in mitochondrial pathology and in the ageing process. Detoxification of mitochondrial superoxide is therefore a promising therapeutic strategy; in addition, a mitochondria-specific superoxide scavenger would be an invaluable laboratory tool.

    We have developed mitoSOD, a mitochondria-targeted version of the manganese superoxide dismutase mimetic M40403 [1]. MitoSOD is directed specifically to mitochondria by the inclusion of the triphenylphosphonium moiety, a lipophilic cation that drives the mitochondrial membrane potential-dependent accumulation of covalently linked compounds within the matrix (to 100-500x the cytosolic concentration) [2]. Previously, TPP-derived compounds have been shown to accumulate within mitochondria of diverse tissues upon oral delivery to mice [3], underscoring their potential utility as therapies for disorders of mitochondrial dysfunction. In this work the antioxidant efficacy of mitoSOD in vitro and within isolated mitochondria was characterised. MitoSOD was shown to compete with cytochrome c for reaction with superoxide, at concentrations comparable to M40403. In addition, mitoSOD competed with NO for reaction with mitochondrial membrane-derived superoxide.

    The study of in vivo mitochondrial superoxide production and detoxification is compromised by the dearth of robust and specific intracellular superoxide probes. Oxidation of dihydroethidium to the red-fluorescent ethidium has been widely used as a measure of intracellular superoxide; however, it is highly susceptible to oxidation in air and is therefore not specific for superoxide. In addition, it does not localise specifically to mitochondria. However, it has recently been suggested that superoxide oxidises dihydroethidium to a fluorescent product that is distinct from ethidium, and is specific for superoxide [4]. We have investigated the feasibility of an HPLC-based assay for mitochondrial superoxide within cells, using mitoSOX, a commercially-available mitochondria-targeted derivative of dihydroethidium.

1.    Salvemini D, Wang Z-Q, Zweier JL, Samouilov A, Macarthur H, Misko TP, Currie MG, Cuzzocrea S, Sikorski JA, Riley DP (1999) A nonpeptidyl mimic of superoxide dismutase with therapeutic activity in rats. Science 286: 304-306.

2.    Ross MF, Kelso GF, Blaikie FH, James AM, Cochemé HM, Filipovska A, Da Ros T, Hurd TR, Smith RAJ, Murphy MP (2005) Biochemistry (Mosc) 70: 222-230.

3.    Smith RA, Porteous CM, Gane AM, Murphy MP (2003) Delivery of bioactive molecules to mitochondria in vivo. PNAS 100: 5407-5412.

4.    Zhao H, Joseph J, Fales HM, Sokoloski EA, Levine RL, Vasquez-Vivar J, Kalyanaraman B (2005) Detection and characterisation of the product of hydroethidine and intracellular superoxide by HPLC and limitations of fluorescence. PNAS 102: 5727-5732.


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