Vanadyl‐induced Fenton‐like reaction in RNA

Abstract

Vanadyl (VO*+) complexed to RNA reacts with hydrogen peroxide in a Fenton-like manner producing hydroxyl radicals ('OH). The hydroxyl radicals can be spin trapped with $5dimethyl-1-pyrroline-l-oxide (DMPO) forming the DMPO-OH spin adduct. In addition, in the presence of ethanol the formation of the hydroxyethyl radical adduct of DMPO (DMPO-ETOH) confirms the production of hydroxyl radicals by the RNA/ VO*' complex. When the reaction between the RNA/VO*+ complex and H202 is carried out in the presence of the spin trap 2-methyL2nitrosopro-pane (MNP), radicals produced in the reaction of 'OH with RNA are trapped. Base hydrolysis of the MNP-RNA adducts (pH 12) followed by a reduction in the pH to pH 7 after hydrolysis is complete, yields an MNP adduct with a well-resolved ESR spectrum identical to the ESR spectrum obtained from analogous experiments with poly U. The ESR spectrum consists of a triplet of sextets (uN = 1.48 mT, a,8=0.25 mT and &=0.14 mT), indicating that the unpaired nitroxide electron interacts with the nuclei of a b-nitrogen and b-hydrogen. The results suggest that the 'OH generated in the RNA/V02+ reaction with H,Oz add to the C(5) carbon of uracil forming a C(6) carbon centered radical. This radical is subsequently spin trapped by MNP.

Tyrosyl radical formation during the oxidative deposition of iron in human apoferritin

“…which is also known to promote Fenton chemistry (Carmichael, 1990) and bind to ferritin (Chasteen & Theil, 1982;Wardeska et al, 1986), likewise produces an H202 spectrum with apoHoSF similar to that seen with Fe2+ in Figure 6. When Fe2+ was added as the EDTA complex using either 02 or H202 as the oxidant (Smith et al, 1990; Tadolini, 1987), only a weak EPR signal unlike those of either Figure 1 or 6 was observed (not shown), implying that Fe2+ binding to specific sites on the apoprotein is a requirement for formation of the HoSF-based radicals seen here.…”

Immunochemical detection of oxidized proteins

“…Bovine serum albumin (BSA)2 was oxidized using a hydroxyl radical generating system consisting of vanadyl and hydrogen peroxide. The vanadium system has been demonstrated to rapidly yield significant quantities of hydroxyl radical at pH 7.4 (15)(16)(17)(18)(19). BSA has been used as a model protein in many oxygen radical studies (4).…”

Oxidative damage during aging targets mitochondrial aconitase

“…BSA was used as the standard for densitometric aconitase carbonyl determination as described by Winn and Wells (27). BSA (1 mg͞ml in 10 mM sodium phosphate, pH 7.4) was oxidized by a hydroxyl radical generating system consisting of 1 mM vanadyl sulfate and 1 mM H 2 O 2 (28)(29)(30)(31). Immediately after the addition of vanadyl sulfate, the protein was derivatized with an equal volume of 0.5 mM DNPH in 0.1 M sodium phosphate buffer (pH 6.3).…”