Iron-induced lipid peroxidation and protein modi®cation in endoplasmic reticulum membranes. Protection by stobadine Peter KaplaÂn*, Michal Doval, Zuzana MajerovaÂ, JaÂn LehotskyÂ, Peter RacÏay Comenius University, Jessenius Faculty of Medicine, Department of Biochemistry, Mala  Hora 4, SK-036 01 Martin, Slovak Republic Received 29 June 1999; accepted 7 December 1999 Abstract Treatment with FeSO 4 /EDTA (0.2 mmol Fe(II) per mg of protein) was used to study the eect of oxidative stress on lipid peroxidation and structural properties of endoplasmic reticulum (ER) membranes isolated from rabbit brain. Oxidative stress resulted in conjugated diene formation and a decrease of 1-anilino-8-naphthalenesulfonate (ANS) ¯uorescence in a time-dependent manner. In contrast, ¯uorescence anisotropy of 1,6-diphenyl-1,3,5- hexatriene was increased early after the initiation of lipid peroxidation and no further increase was observed after 1, 2 and 3 h of peroxidation. FeSO 4 /EDTA treatment was accompanied by formation of conjugates of lipid peroxidation products with membrane proteins, as detected by the increase in ¯uorescence excitation (350±360 nm) and emission (440±450 nm) maximum. Oxidative stress also induced a marked decrease of the intrinsic ¯uorescence of aromatic amino acids, suggesting modi®cation or changes in the environment of these amino acid residue(s). The lipid antioxidant, stobadine, completely prevented the changes of ANS ¯uorescence and production of peroxidized lipid±protein conjugates whereas tryptophan ¯uorescence was only partially protected. These results suggest that Fe(II) induces both lipid-mediated- and lipid peroxidation independent-modi®cation of ER membrane proteins. The study also demonstrates that stobadine is a potent inhibitor of Fe(II)-induced protein modi®cation. 7 2000 Elsevier Science Ltd. All rights reserved. Keywords: Free radical; Iron; Endoplasmic reticulum; Antioxidant; Fluorescence 1. Introduction Several neurological disorders including brain ischemia and Alzheimer's disease are associated with the perturbation of intracellular calcium concentration [Ca 2+ ] i and stimulation of oxygen free radical formation [1±3]. Although these ®nd- ings suggest that free radicals may play an im- 1357-2725/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved. PII: S1357-2725(99)00147-8 * Corresponding author. Tel.: +421-842-41315-65; fax: +421-842-41367-70. E-mail address: kaplan@jfmed.uniba.sk (P. KaplaÂn). Abbreviations: ANS, 1-anilino-8-naphthalenesulfonate; DPH, 1; 6-diphenyl-1,3,5-hexatriene; EDTA, Ethylenediamine tetraacetic acid; ER, Endoplasmic reticulum; SERCA, Sarco/ endoplasmic reticulum Ca 2+ -transport ATPase; SR, Sarco- plasmic reticulum.