Reexamination of the mechanism of hydroxyl radical adducts formed from the reaction between familial amyotrophic lateral sclerosis-associated Cu,Zn superoxide dismutase mutants and H2O2

Ravinder Jit Singh, H. Karoui, M. R. Gunther, J. S. Beckman, R. P. Mason, B. Kalyanaraman

Research output: Contribution to journalArticle

104 Citations (Scopus)

Abstract

Amyotrophic lateral sclerosis (ALS) involves the progressive degeneration of motor neurons in the spinal cord and motor cortex. Mutations to Cu,Zn superoxide dismutase (SOD) linked with familial ALS are reported to increase hydroxyl radical adduct formation from hydrogen peroxide as measured by spin trapping with 5,5'-dimethyl-1-pyrrolline N-oxide (DMPO). In the present study, we have used oxygen-17-enriched water and H2O2 to reinvestigate the mechanism of DMPO/·OH formation from the SOD and SOD mutants. The relative ratios of DMPO/(·17)OH and DMPO/(·16)OH formed in the Fenton reaction were 90% and 10%, respectively, reflecting the ratios of H2 17O2 to H2 16O2. The reaction of the WT SOD with H2 17O2 in bicarbonate/CO2 buffer yielded 63% DMPO/(·17)OH and 37% DMPO/(·16)OH. Similar results were obtained from the reaction between familial ALS SOD mutants and H2 17O2: DMPO/(·17)OH (64%); DMPO/(·16)OH (36%) from A4V and DMPO/(·17)OH (62%); and DMPO/(·16)OH (38%) from G93A. These results were confirmed further by using 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide spin trap, a phosphorylated analog of DMPO. Contrary to earlier reports, the present results indicate that a significant fraction of DMPO/·OH formed during the reaction of SOD and familial ALS SOD mutants with H2O2 is derived from the incorporation of oxygen from water due to oxidation of DMPO to DMPO/·OH presumably via DMPO radical cation. No differences were detected between WT and mutant SODs, neither in the concentration of DMPO/·OH or DEPMPO/·OH formed nor in the relative incorporation of oxygen from H2O2 or water.

Original languageEnglish (US)
Pages (from-to)6675-6680
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume95
Issue number12
DOIs
StatePublished - Jun 9 1998

Fingerprint

Hydroxyl Radical
Oxides
Superoxide Dismutase
Superoxide Dismutase-1
Amyotrophic lateral sclerosis 1
Oxygen
Water
Spin Trapping
Amyotrophic Lateral Sclerosis
Motor Cortex
Motor Neurons
Bicarbonates
Hydrogen Peroxide
Cations
Spinal Cord
Buffers

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

@article{636d76aecc90440ebbd5b996cbdd8079,
title = "Reexamination of the mechanism of hydroxyl radical adducts formed from the reaction between familial amyotrophic lateral sclerosis-associated Cu,Zn superoxide dismutase mutants and H2O2",
abstract = "Amyotrophic lateral sclerosis (ALS) involves the progressive degeneration of motor neurons in the spinal cord and motor cortex. Mutations to Cu,Zn superoxide dismutase (SOD) linked with familial ALS are reported to increase hydroxyl radical adduct formation from hydrogen peroxide as measured by spin trapping with 5,5'-dimethyl-1-pyrrolline N-oxide (DMPO). In the present study, we have used oxygen-17-enriched water and H2O2 to reinvestigate the mechanism of DMPO/·OH formation from the SOD and SOD mutants. The relative ratios of DMPO/(·17)OH and DMPO/(·16)OH formed in the Fenton reaction were 90{\%} and 10{\%}, respectively, reflecting the ratios of H2 17O2 to H2 16O2. The reaction of the WT SOD with H2 17O2 in bicarbonate/CO2 buffer yielded 63{\%} DMPO/(·17)OH and 37{\%} DMPO/(·16)OH. Similar results were obtained from the reaction between familial ALS SOD mutants and H2 17O2: DMPO/(·17)OH (64{\%}); DMPO/(·16)OH (36{\%}) from A4V and DMPO/(·17)OH (62{\%}); and DMPO/(·16)OH (38{\%}) from G93A. These results were confirmed further by using 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide spin trap, a phosphorylated analog of DMPO. Contrary to earlier reports, the present results indicate that a significant fraction of DMPO/·OH formed during the reaction of SOD and familial ALS SOD mutants with H2O2 is derived from the incorporation of oxygen from water due to oxidation of DMPO to DMPO/·OH presumably via DMPO radical cation. No differences were detected between WT and mutant SODs, neither in the concentration of DMPO/·OH or DEPMPO/·OH formed nor in the relative incorporation of oxygen from H2O2 or water.",
author = "Singh, {Ravinder Jit} and H. Karoui and Gunther, {M. R.} and Beckman, {J. S.} and Mason, {R. P.} and B. Kalyanaraman",
year = "1998",
month = "6",
day = "9",
doi = "10.1073/pnas.95.12.6675",
language = "English (US)",
volume = "95",
pages = "6675--6680",
journal = "Proceedings of the National Academy of Sciences of the United States of America",
issn = "0027-8424",
number = "12",

}

TY - JOUR

T1 - Reexamination of the mechanism of hydroxyl radical adducts formed from the reaction between familial amyotrophic lateral sclerosis-associated Cu,Zn superoxide dismutase mutants and H2O2

AU - Singh, Ravinder Jit

AU - Karoui, H.

AU - Gunther, M. R.

AU - Beckman, J. S.

AU - Mason, R. P.

AU - Kalyanaraman, B.

PY - 1998/6/9

Y1 - 1998/6/9

N2 - Amyotrophic lateral sclerosis (ALS) involves the progressive degeneration of motor neurons in the spinal cord and motor cortex. Mutations to Cu,Zn superoxide dismutase (SOD) linked with familial ALS are reported to increase hydroxyl radical adduct formation from hydrogen peroxide as measured by spin trapping with 5,5'-dimethyl-1-pyrrolline N-oxide (DMPO). In the present study, we have used oxygen-17-enriched water and H2O2 to reinvestigate the mechanism of DMPO/·OH formation from the SOD and SOD mutants. The relative ratios of DMPO/(·17)OH and DMPO/(·16)OH formed in the Fenton reaction were 90% and 10%, respectively, reflecting the ratios of H2 17O2 to H2 16O2. The reaction of the WT SOD with H2 17O2 in bicarbonate/CO2 buffer yielded 63% DMPO/(·17)OH and 37% DMPO/(·16)OH. Similar results were obtained from the reaction between familial ALS SOD mutants and H2 17O2: DMPO/(·17)OH (64%); DMPO/(·16)OH (36%) from A4V and DMPO/(·17)OH (62%); and DMPO/(·16)OH (38%) from G93A. These results were confirmed further by using 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide spin trap, a phosphorylated analog of DMPO. Contrary to earlier reports, the present results indicate that a significant fraction of DMPO/·OH formed during the reaction of SOD and familial ALS SOD mutants with H2O2 is derived from the incorporation of oxygen from water due to oxidation of DMPO to DMPO/·OH presumably via DMPO radical cation. No differences were detected between WT and mutant SODs, neither in the concentration of DMPO/·OH or DEPMPO/·OH formed nor in the relative incorporation of oxygen from H2O2 or water.

AB - Amyotrophic lateral sclerosis (ALS) involves the progressive degeneration of motor neurons in the spinal cord and motor cortex. Mutations to Cu,Zn superoxide dismutase (SOD) linked with familial ALS are reported to increase hydroxyl radical adduct formation from hydrogen peroxide as measured by spin trapping with 5,5'-dimethyl-1-pyrrolline N-oxide (DMPO). In the present study, we have used oxygen-17-enriched water and H2O2 to reinvestigate the mechanism of DMPO/·OH formation from the SOD and SOD mutants. The relative ratios of DMPO/(·17)OH and DMPO/(·16)OH formed in the Fenton reaction were 90% and 10%, respectively, reflecting the ratios of H2 17O2 to H2 16O2. The reaction of the WT SOD with H2 17O2 in bicarbonate/CO2 buffer yielded 63% DMPO/(·17)OH and 37% DMPO/(·16)OH. Similar results were obtained from the reaction between familial ALS SOD mutants and H2 17O2: DMPO/(·17)OH (64%); DMPO/(·16)OH (36%) from A4V and DMPO/(·17)OH (62%); and DMPO/(·16)OH (38%) from G93A. These results were confirmed further by using 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide spin trap, a phosphorylated analog of DMPO. Contrary to earlier reports, the present results indicate that a significant fraction of DMPO/·OH formed during the reaction of SOD and familial ALS SOD mutants with H2O2 is derived from the incorporation of oxygen from water due to oxidation of DMPO to DMPO/·OH presumably via DMPO radical cation. No differences were detected between WT and mutant SODs, neither in the concentration of DMPO/·OH or DEPMPO/·OH formed nor in the relative incorporation of oxygen from H2O2 or water.

UR - http://www.scopus.com/inward/record.url?scp=0032499788&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0032499788&partnerID=8YFLogxK

U2 - 10.1073/pnas.95.12.6675

DO - 10.1073/pnas.95.12.6675

M3 - Article

C2 - 9618471

AN - SCOPUS:0032499788

VL - 95

SP - 6675

EP - 6680

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

SN - 0027-8424

IS - 12

ER -