PMID- 14724284
OWN - NLM
STAT- MEDLINE
DCOM- 20040507
LR  - 20240109
IS  - 0021-9258 (Print)
IS  - 0021-9258 (Linking)
VI  - 279
IP  - 13
DP  - 2004 Mar 26
TI  - Protein radical formation during lactoperoxidase-mediated oxidation of the 
      suicide substrate glutathione: immunochemical detection of a lactoperoxidase 
      radical-derived 5,5-dimethyl-1-pyrroline N-oxide nitrone adduct.
PG  - 13272-83
AB  - 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.
FAU - Guo, Qiong
AU  - Guo Q
AD  - Laboratory of Pharmacology and Chemistry, National Institute of Environmental 
      Health Sciences, National Institutes of Health, PO Box 12233, Research Triangle 
      Park, NC 27709, USA. guo1@niehs.nih.gov
FAU - Detweiler, Charles D
AU  - Detweiler CD
FAU - Mason, Ronald P
AU  - Mason RP
LA  - eng
PT  - Journal Article
PT  - Research Support, U.S. Gov't, P.H.S.
DEP - 20040114
PL  - United States
TA  - J Biol Chem
JT  - The Journal of biological chemistry
JID - 2985121R
RN  - 0 (Amino Acids)
RN  - 0 (Azides)
RN  - 0 (Cyclic N-Oxides)
RN  - 0 (Free Radicals)
RN  - 0 (Iodides)
RN  - 0 (Nitrites)
RN  - 0 (Nitrogen Oxides)
RN  - 0 (Spin Labels)
RN  - 0 (Thiocyanates)
RN  - 0 (nitrones)
RN  - 339NCG44TV (Phenol)
RN  - 42HK56048U (Tyrosine)
RN  - 42VZT0U6YR (Heme)
RN  - 58134-17-1 (5,5-dimethyl-2-phenyl-1-pyrroline-N-oxide)
RN  - BBX060AN9V (Hydrogen Peroxide)
RN  - EC 1.11.1.- (Lactoperoxidase)
RN  - EC 1.11.1.6 (Catalase)
RN  - GAN16C9B8O (Glutathione)
RN  - K848JZ4886 (Cysteine)
RN  - O748SU14OM (thiocyanate)
RN  - PQ6CK8PD0R (Ascorbic Acid)
RN  - S88TT14065 (Oxygen)
SB  - IM
MH  - Amino Acids/chemistry
MH  - Ascorbic Acid/pharmacology
MH  - Azides/pharmacology
MH  - Blotting, Western
MH  - Catalase/pharmacology
MH  - Cyclic N-Oxides/chemistry
MH  - Cysteine/chemistry
MH  - Dose-Response Relationship, Drug
MH  - Electron Spin Resonance Spectroscopy
MH  - Electrophoresis, Polyacrylamide Gel
MH  - Enzyme-Linked Immunosorbent Assay
MH  - Free Radicals
MH  - Glutathione/*chemistry/metabolism
MH  - Heme/chemistry
MH  - Hydrogen Peroxide/pharmacology
MH  - Hydrogen-Ion Concentration
MH  - Immunochemistry
MH  - Iodides/pharmacology
MH  - Lactoperoxidase/*chemistry
MH  - Models, Chemical
MH  - Nitrites/metabolism/pharmacology
MH  - Nitrogen Oxides
MH  - Oxygen/*metabolism
MH  - Oxygen Consumption
MH  - Phenol/pharmacology
MH  - Spin Labels
MH  - Thiocyanates/pharmacology
MH  - Time Factors
MH  - Tyrosine/chemistry
EDAT- 2004/01/16 05:00
MHDA- 2004/05/08 05:00
CRDT- 2004/01/16 05:00
PHST- 2004/01/16 05:00 [pubmed]
PHST- 2004/05/08 05:00 [medline]
PHST- 2004/01/16 05:00 [entrez]
AID - S0021-9258(19)64277-8 [pii]
AID - 10.1074/jbc.M310034200 [doi]
PST - ppublish
SO  - J Biol Chem. 2004 Mar 26;279(13):13272-83. doi: 10.1074/jbc.M310034200. Epub 2004 
      Jan 14.