Different cellular targets for Cu- and Fe-catalyzed oxidation observed using a Cu-compatible thiobarbituric acid assay

• Published in: Biochimica et biophysica acta. General subjects Vol. 1035; no. 3; pp. 353 - 360
• Main Authors: Gelvan, Dan, Saltman, Paul
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 01.09.1990
• Subjects: SDS; TBARS; MDA; EDTA; Tris; TBA; RBC; TCA; TMP; Hct; Hb; NTA; GDW
• ISSN: 0304-4165; 1872-8006
• DOI: 10.1016/0304-4165(90)90100-B

The widely used thiobarbituric acid (TBA) assay for oxidative damage to biomolecules fails in Cu 2+ -containing solutions due to the formation of a cloudy precipitate. The chelatin of Cu 2+ ions with EDTA or Chelex was investigated. Both prevented precipitate formation, but only Chelex allowed proper color development in the TBA assay. The Chelex modified assay could be adapted to a variety of systems, and was applied to the detection of Cu 2+/ascorbate dependent deoxyribose breakdown and oxidative damage in erythrocyte ghosts, lysates and whole cells. Using this method, it was shown that Cu 2+/ascorbate caused membrane damage in ghosts but not in whole red blood cells (RBC). Fe 3+/ascorbate, on the other hand, caused formation of TBA-reactive products even in whole RBC. When Cu 2+ and Fe 3+ were presented to isolated hemoglobin as their 1:1 nitrilotriacetate complexes, the protein bound 10–12 cupric ions per molecule, but no ferric ions. It is suggested that oxidative damage catalyzed by copper or iron ions has different cellular targets, determined by the different binding properties of the two metals to various cellular components.