On-line EPR study of free radicals induced by peroxidase/H(2)O(2) in human low-density lipoprotein.
The aim of this study was to use direct electron paramagnetic resonance (EPR) spectroscopy at 37 degrees C and spin trapping techniques to study radical species formed during horseradish peroxidase/H(2)O(2)-initiated low-density lipoprotein (LDL) oxidation. Using direct EPR, we obtained evidence for the formation not only of the alpha-tocopheroxyl radical but also of a protein radical(s), assigned to a tyrosyl radical(s) of apolipoprotein B-100 (apo B-100). Spin trapping with 2-methyl-2-nitrosopropane revealed (i) the formation of a mobile adduct with beta-hydrogen coupling assigned to a lipid radical and (ii) a partially immobilised adduct detected in LDL as well as in apo B-100, assigned after proteolytic digestion to the trapping of a radical centred on a tertiary carbon atom of an aromatic residue, probably tyrosine. Our results support the hypothesis that radicals are initiators of the oxidative process, and show that their formation is an early event in peroxidase-mediated oxidation. We also tested the effects of resveratrol (RSV), a polyphenolic antioxidant present in red wine. Our data indicate that 1-10 microM RSV is able to accelerate alpha-tocopherol consumption, conjugated dienes formation and the decay kinetics of LDL-centred radicals. Since phenols are substrates for peroxidases, this result may be ascribed to a RSV-mediated catalysis of peroxidase activity.
Pietraforte
Donatella
D
Laboratorio di Biologia Cellulare, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161, Rome, Italy. pietraf@iss.it
Turco
Laura
L
Azzini
Elena
E
Minetti
Maurizio
M
D000975:Antioxidants / Q000494:pharmacology*; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals; D006735:Horseradish Peroxidase / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism* / Q000494:pharmacology; D008077:Lipoproteins, LDL / Q000378:metabolism*; D009603:Nitroso Compounds; D016877:Oxidants / Q000378:metabolism* / Q000494:pharmacology; D000077185:Resveratrol; D019097:Spin Trapping; D013267:Stilbenes / Q000494:pharmacology*; D013696:Temperature
D000975:Antioxidants; D005609:Free Radicals; D008077:Lipoproteins, LDL; D009603:Nitroso Compounds; D016877:Oxidants; D013267:Stilbenes; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase; C034526:tert-nitrosobutane; D000077185:Resveratrol