Original Contribution MEASUREMENTS OF PROTEIN CARBONYLS, ORTHO- AND META- TYROSINE AND OXIDATIVE PHOSPHORYLATION COMPLEX ACTIVITY IN MITOCHONDRIA FROM YOUNG AND OLD RATS STEFAN M. K. DAVIES, 1 *ANNE POLJAK, 1² MARK W. DUNCAN, ‡ GEORGE A. SMYTHE, ² and MICHAEL P. MURPHY § *Department of Biochemistry, University of Otago, Dunedin, New Zealand; ² Ray Williams Biomedical Mass Spectrometry Facility, University of New South Wales, Sydney, Australia; ‡ Department of Pharmaceutical Sciences, University of Colorado Health Sciences Center, Denver, CO, USA; and § MRC-Dunn Human Nutrition Unit, Cambridge, UK (Received 13 February 2001; Accepted 19 April 2001) Abstract—Mitochondrial bioenergetic function is often reported to decline with age and the accumulation of oxidative damage is thought to contribute. However, there are considerable uncertainties about the amount and significance of mitochondrial oxidative damage in aging. We hypothesized that, as radical production in mitochondria is greater than the rest of the cell, protein oxidative damage should accumulate more in mitochondria than the cytoplasm, and that this relative accumulation should increase with age. To test these hypotheses we measured the accumulation of three markers of protein oxidative damage in liver, brain, and heart from young and old rats. Ortho- and meta-tyrosine levels in protein hydrolysates were measured by a gas chromatography/mass spectrometry assay, and protein carbonyl content was determined by ELISA. Using these assays we found no evidence for increased protein oxidative damage in mitochondria relative to the cytosol. Most increases found in protein oxidative damage on aging were modest for all three tissues and there was no consistent pattern of increased oxidative damage in mitochondrial proteins on aging. Mitochondrial oxidative phosphorylation complex activities were also assessed revealing 39 – 42% decreases in F 0 F 1 - ATP synthase activity in liver and heart on aging, but not in other oxidative phosphorylation complexes. These findings have implications for the contribution of mitochondrial oxidative damage and dysfunction to aging. © 2001 Elsevier Science Inc. Keywords—Mitochondria, Protein oxidative damage, Aging, Oxidative phosphorylation complexes, Free radicals INTRODUCTION The role and origin of mitochondrial dysfunction in aging is of considerable interest, but while there is a range of evidence that mitochondrial bioenergetic func- tion declines with aging [1], the mechanism and func- tional significance of this decline are uncertain [2– 8]. Cumulative oxidative damage is often proposed as the major cause of mitochondrial dysfunction with aging, because the respiratory chain is a major source of dam- aging free radicals [8,9]. Mitochondrial dysfunction could arise from oxidative damage by a number of mech- anisms such as damage to mitochondrial DNA (mtDNA) leading to mutations, insertions or deletions that cause the defective assembly of oxidative phosphorylation complexes; lipid peroxidation increasing the proton per- meability of the mitochondrial inner membrane, or pro- tein oxidative damage disrupting critical enzymes or transporters [8,10 –13]. A role for mitochondrial oxida- tive damage in aging is supported by reports of increased mitochondrial lipid peroxidation, accumulation of oxi- dized DNA bases, generalized protein oxidative damage, and increased oxidative damage to specific proteins in mitochondria on aging [10,12–15]. However, some ap- proaches to measuring oxidative damage are nonspecific and prone to interference [16 –18], and there are also reports of no change in oxidative damage with aging [16,19]. Therefore, the amount and significance of cu- mulative mitochondrial oxidative damage in aging is Address correspondence to: Dr. Michael P. Murphy, MRC-Dunn Human Nutrition Unit, Wellcome Trust-MRC Building, Hills Road, Cambridge, CB2 2XY, UK; Fax: +44 (1223) 252705; E-Mail: mpm@mrc-dunn.cam.ac.uk. 1 These authors contributed equally to this work. Free Radical Biology & Medicine, Vol. 31, No. 2, pp. 181–190, 2001 Copyright © 2001 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/01/$–see front matter PII S0891-5849(01)00576-7 181