Protein Radical Formation during Lactoperoxidase-mediated Oxidation of the Suicide Substrate Glutathione

Abstract

A novel anti-5,5-dimethyl-1-pyrroline N-oxide (DMPO) polyclonal antiserum that specifically recognizes protein radical-derived DMPO nitrone adducts has been developed. In this study, we employed this new approach, which combines the specificity of spin trapping and the sensitivity of antigen-antibody interactions, to investigate protein radical formation from lactoperoxidase (LPO). When LPO reacted with GSH in the presence of DMPO, we detected an LPO radical-derived DMPO nitrone adduct using enzyme-linked immunosorbent assay and Western blotting. The formation of this nitrone adduct depended on the concentrations of GSH, LPO, and DMPO as well as pH values, and GSH could not be replaced by H 2 O 2 . The level of this nitrone adduct was decreased significantly by azide, catalase, ascorbate, iodide, thiocyanate, phenol, or nitrite. However, its formation was unaffected by chemical modification of free cysteine, tyrosine, and tryptophan residues on LPO. ESR spectra showed that a glutathiyl radical was formed from the LPO/GSH/DMPO system, but no protein radical adduct could be detected by ESR. Its formation was decreased by azide, catalase, ascorbate, iodide, or thiocyanate, whereas phenol or nitrite increased it. GSH caused marked changes in the spectrum of compound II of LPO, indicating that GSH binds to the heme of compound II, whereas phenol or nitrite prevented these changes and reduced compound II back to the native enzyme. GSH also dose-dependently inhibited the peroxidase activity of LPO as determined by measuring 2,2 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) oxidation. Taken together, these results demonstrate that the GSH-dependent LPO radical formation is mediated by the glutathiyl radical, possibly via the reaction of the glutathiyl radical with the heme of compound II to form a heme-centered radical trapped by DMPO.

C-Phycocyanin Confers Protection against Oxalate-Mediated Oxidative Stress and Mitochondrial Dysfunctions in MDCK Cells

“…Free radicals such as H 2 O 2 and superoxide anions are potent mediators of onset of LPO [43]. These effectors reportedly disconcert membrane integrity leading to the release of a plethora of mitochondrial activators of the cytosolic cysteinyl aspartate proteases (caspases) [44].…”

Two New Nitrone Alkaloids from Huperzia serrata

“…-The family of nitrone compounds is a kind of spin-trapping composition, which reacts with the free radical to form a compound called a spin adduct. Once the adduct is formed, it is relatively stable, and the radical thus becomes inactivated and unable to damage cellular tissues or biochemical processes [1] [2]. Studies have reported that the nitrone spin trap a-phenyl tert-butyl nitrone can significantly ameliorate neuronal cell loss and neurologic deficits induced by stroke in a gerbil model of global ischemia [3].…”

Identification of free radicals in oxidized and glycoxidized phosphatidylethanolamines by spin trapping combined with tandem mass spectrometry

“…It is known that spin‐trapping techniques can provide direct evidence of the presence of intermediate radical species in the reaction system. The combined use of spin trapping and mass spectrometry (MS) is a sensitive technique that has been used by a number of groups for identifying radical species of amino acids, peptides, proteins, lipids, and phosphatidylcholines . In the present work, radical intermediaries formed during the oxidation of palmitoyl‐lineoyl phosphatidylethanolmine (PLPE) and glycated PLPE (gPLPE) (Scheme ) were trapped using a spin trap (DMPO) and the radical adducts formed were analyzed by electrospray ionization tandem mass spectrometry (ESI‐MS/MS).…”