tailieunhanh - Báo cáo khoa học: Hydrogen peroxide efflux from muscle mitochondria underestimates matrix superoxide production – a correction using glutathione depletion

The production of H2O2 by isolated mitochondria is frequently used as a measure of mitochondrial superoxide formation. Matrix superoxide dismu-tase quantitatively converts matrix superoxide to H2O2. However, matrix enzymes such as the glutathione peroxidases can consume H2O2 and com-pete with efflux of H2O2 , causing an underestimation of superoxide produc-tion. | ễFEBS Journal Hydrogen peroxide efflux from muscle mitochondria underestimates matrix superoxide production - a correction using glutathione depletion Jason R. Treberg Casey L. Quinlan and Martin D. Brand Buck Institute for Age Research Novato CA USA Keywords 1-chloro-2 4-dinitrobenzene complex I complex III peroxidase reactive oxygen species Correspondence J. R. Treberg Buck Institute for Age Research 8001 Redwood Boulevard Novato CA 94945 USA Fax 415 209 2232 Tel 415 209 2000 E-mail jtreberg@ Received 15 February 2010 revised 31 March 2010 accepted 22 April 2010 doi The production of H2O2 by isolated mitochondria is frequently used as a measure of mitochondrial superoxide formation. Matrix superoxide dismutase quantitatively converts matrix superoxide to H2O2. However matrix enzymes such as the glutathione peroxidases can consume H2O2 and compete with efflux of H2O2 causing an underestimation of superoxide production. To assess this underestimate we depleted matrix glutathione in rat skeletal muscle mitochondria by more than 90 as a consequence of pretreatment with 1-chloro-2 4-dintrobenzene CDNB . The pretreatment protocol strongly diminished the mitochondrial capacity to consume exogenous H2O2 consistent with decreased peroxidase capacity but avoided direct stimulation of superoxide production from complex I. It elevated the observed rates of H2O2 formation from matrix-directed superoxide by up to two-fold from several sites of production as defined by substrates and electron transport inhibitors over a wide range of control rates from nmol H2O2-min-1-mg protein-1. Similar results were obtained when glutathione was depleted using monochlorobimane or when soluble matrix peroxidase activity was removed by preparation of submito-chondrial particles. The data indicate that the increased H2O2 efflux observed with CDNB pretreatment was a result of glutathione depletion and compromised peroxidase activity. A hyperbolic .