Protein sulfhydryls are protected from irreversible oxidation by conversion to mixed disulfides

Carol Coan, Ji Ying Ji, K. Hideg, Rolf J. Mehlhorn

Research output: Contribution to journalArticle

60 Citations (Scopus)

Abstract

Protein mixed thioselenides formed by reaction of sarcoplasmic reticulum (SR) with diselenide biradical spin labels were quantified by ESR. Whereas the reaction of SR membranes with the diselenide spin label led to a large ESR signal of the unbound monoselenide at equilibrium, treatment of the reaction mixture with a few millimolar hydrogen peroxide converted all of the nitroxides to protein-bound thioselenides. This technique of spin-labeling protein thiols avoids the need to remove unreacted spin labels. The bound spin labels were removable by reduction with excess mercaptoethanol, indicating a specific and reversible labeling of protein thiols. SR that had been extensively labeled with the diselenide spin label was resistant to ATPase inactivation by potent oxidants that arise when myoglobin reacts with hydroperoxides. Unmodified SR lost all activity within 10 min of exposure to either 1 mm tert-butyl hydroperoxide in the presence of 200 μm equine myoglobin or to 100 mm hydrogen peroxide in the absence of myoglobin. In both cases the loss of activity could not be reversed by subsequent treatment with mercaptoethanol. On the other hand, membranes that had been extensively treated with the diselenide spin label and were then subjected to these peroxide treatments were fully active after mercaptoethanol-mediated cleavage of the thioselenides. ESR analysis of spin-labeled SR showed no detectable oxidative cleavage of the thioselenide bonds. Sodium dodecyl sulfate gel electrophoresis showed that peroxide-mediated crosslinking of ATPase observed in unmodified SR membranes did not occur in the diselenide-modified SR membranes. Only limited protection was observed when SR pretreated with glutathione disulfide was incubated with hydroperoxides. In this case, however, the degree of protection was greatly increased when the reaction with glutathione disulfide was carried out in the presence of the supernatant of centrifuged rat liver homogenate, consistent with an acceleration of mixed disulfide formation by a factor tentatively identified as thiol transferase. It is concluded that conversion of protein thiol residues to either thioselenides or mixed disulfides confers protection against irreversible peroxide-dependent oxidation. We suggest that mixed disulfide formation by thiol transferase activity may help protect protein thiols from irreversible oxidation by heme-activated hydroperoxides.

Original languageEnglish
Pages (from-to)369-378
Number of pages10
JournalArchives of Biochemistry and Biophysics
Volume295
Issue number2
DOIs
Publication statusPublished - 1992

Fingerprint

Spin Labels
Sarcoplasmic Reticulum
Sulfhydryl Compounds
Disulfides
Hydrogen Peroxide
Oxidation
Mercaptoethanol
Myoglobin
Peroxides
Membranes
Paramagnetic resonance
Proteins
Glutathione Disulfide
Transferases
Labeling
Adenosine Triphosphatases
tert-Butylhydroperoxide
Electrophoresis
Heme
Oxidants

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Protein sulfhydryls are protected from irreversible oxidation by conversion to mixed disulfides. / Coan, Carol; Ji, Ji Ying; Hideg, K.; Mehlhorn, Rolf J.

In: Archives of Biochemistry and Biophysics, Vol. 295, No. 2, 1992, p. 369-378.

Research output: Contribution to journalArticle

@article{cf8750d892034acdb63dbca8a0f1b3a6,
title = "Protein sulfhydryls are protected from irreversible oxidation by conversion to mixed disulfides",
abstract = "Protein mixed thioselenides formed by reaction of sarcoplasmic reticulum (SR) with diselenide biradical spin labels were quantified by ESR. Whereas the reaction of SR membranes with the diselenide spin label led to a large ESR signal of the unbound monoselenide at equilibrium, treatment of the reaction mixture with a few millimolar hydrogen peroxide converted all of the nitroxides to protein-bound thioselenides. This technique of spin-labeling protein thiols avoids the need to remove unreacted spin labels. The bound spin labels were removable by reduction with excess mercaptoethanol, indicating a specific and reversible labeling of protein thiols. SR that had been extensively labeled with the diselenide spin label was resistant to ATPase inactivation by potent oxidants that arise when myoglobin reacts with hydroperoxides. Unmodified SR lost all activity within 10 min of exposure to either 1 mm tert-butyl hydroperoxide in the presence of 200 μm equine myoglobin or to 100 mm hydrogen peroxide in the absence of myoglobin. In both cases the loss of activity could not be reversed by subsequent treatment with mercaptoethanol. On the other hand, membranes that had been extensively treated with the diselenide spin label and were then subjected to these peroxide treatments were fully active after mercaptoethanol-mediated cleavage of the thioselenides. ESR analysis of spin-labeled SR showed no detectable oxidative cleavage of the thioselenide bonds. Sodium dodecyl sulfate gel electrophoresis showed that peroxide-mediated crosslinking of ATPase observed in unmodified SR membranes did not occur in the diselenide-modified SR membranes. Only limited protection was observed when SR pretreated with glutathione disulfide was incubated with hydroperoxides. In this case, however, the degree of protection was greatly increased when the reaction with glutathione disulfide was carried out in the presence of the supernatant of centrifuged rat liver homogenate, consistent with an acceleration of mixed disulfide formation by a factor tentatively identified as thiol transferase. It is concluded that conversion of protein thiol residues to either thioselenides or mixed disulfides confers protection against irreversible peroxide-dependent oxidation. We suggest that mixed disulfide formation by thiol transferase activity may help protect protein thiols from irreversible oxidation by heme-activated hydroperoxides.",
author = "Carol Coan and Ji, {Ji Ying} and K. Hideg and Mehlhorn, {Rolf J.}",
year = "1992",
doi = "10.1016/0003-9861(92)90530-A",
language = "English",
volume = "295",
pages = "369--378",
journal = "Archives of Biochemistry and Biophysics",
issn = "0003-9861",
publisher = "Academic Press Inc.",
number = "2",

}

TY - JOUR

T1 - Protein sulfhydryls are protected from irreversible oxidation by conversion to mixed disulfides

AU - Coan, Carol

AU - Ji, Ji Ying

AU - Hideg, K.

AU - Mehlhorn, Rolf J.

PY - 1992

Y1 - 1992

N2 - Protein mixed thioselenides formed by reaction of sarcoplasmic reticulum (SR) with diselenide biradical spin labels were quantified by ESR. Whereas the reaction of SR membranes with the diselenide spin label led to a large ESR signal of the unbound monoselenide at equilibrium, treatment of the reaction mixture with a few millimolar hydrogen peroxide converted all of the nitroxides to protein-bound thioselenides. This technique of spin-labeling protein thiols avoids the need to remove unreacted spin labels. The bound spin labels were removable by reduction with excess mercaptoethanol, indicating a specific and reversible labeling of protein thiols. SR that had been extensively labeled with the diselenide spin label was resistant to ATPase inactivation by potent oxidants that arise when myoglobin reacts with hydroperoxides. Unmodified SR lost all activity within 10 min of exposure to either 1 mm tert-butyl hydroperoxide in the presence of 200 μm equine myoglobin or to 100 mm hydrogen peroxide in the absence of myoglobin. In both cases the loss of activity could not be reversed by subsequent treatment with mercaptoethanol. On the other hand, membranes that had been extensively treated with the diselenide spin label and were then subjected to these peroxide treatments were fully active after mercaptoethanol-mediated cleavage of the thioselenides. ESR analysis of spin-labeled SR showed no detectable oxidative cleavage of the thioselenide bonds. Sodium dodecyl sulfate gel electrophoresis showed that peroxide-mediated crosslinking of ATPase observed in unmodified SR membranes did not occur in the diselenide-modified SR membranes. Only limited protection was observed when SR pretreated with glutathione disulfide was incubated with hydroperoxides. In this case, however, the degree of protection was greatly increased when the reaction with glutathione disulfide was carried out in the presence of the supernatant of centrifuged rat liver homogenate, consistent with an acceleration of mixed disulfide formation by a factor tentatively identified as thiol transferase. It is concluded that conversion of protein thiol residues to either thioselenides or mixed disulfides confers protection against irreversible peroxide-dependent oxidation. We suggest that mixed disulfide formation by thiol transferase activity may help protect protein thiols from irreversible oxidation by heme-activated hydroperoxides.

AB - Protein mixed thioselenides formed by reaction of sarcoplasmic reticulum (SR) with diselenide biradical spin labels were quantified by ESR. Whereas the reaction of SR membranes with the diselenide spin label led to a large ESR signal of the unbound monoselenide at equilibrium, treatment of the reaction mixture with a few millimolar hydrogen peroxide converted all of the nitroxides to protein-bound thioselenides. This technique of spin-labeling protein thiols avoids the need to remove unreacted spin labels. The bound spin labels were removable by reduction with excess mercaptoethanol, indicating a specific and reversible labeling of protein thiols. SR that had been extensively labeled with the diselenide spin label was resistant to ATPase inactivation by potent oxidants that arise when myoglobin reacts with hydroperoxides. Unmodified SR lost all activity within 10 min of exposure to either 1 mm tert-butyl hydroperoxide in the presence of 200 μm equine myoglobin or to 100 mm hydrogen peroxide in the absence of myoglobin. In both cases the loss of activity could not be reversed by subsequent treatment with mercaptoethanol. On the other hand, membranes that had been extensively treated with the diselenide spin label and were then subjected to these peroxide treatments were fully active after mercaptoethanol-mediated cleavage of the thioselenides. ESR analysis of spin-labeled SR showed no detectable oxidative cleavage of the thioselenide bonds. Sodium dodecyl sulfate gel electrophoresis showed that peroxide-mediated crosslinking of ATPase observed in unmodified SR membranes did not occur in the diselenide-modified SR membranes. Only limited protection was observed when SR pretreated with glutathione disulfide was incubated with hydroperoxides. In this case, however, the degree of protection was greatly increased when the reaction with glutathione disulfide was carried out in the presence of the supernatant of centrifuged rat liver homogenate, consistent with an acceleration of mixed disulfide formation by a factor tentatively identified as thiol transferase. It is concluded that conversion of protein thiol residues to either thioselenides or mixed disulfides confers protection against irreversible peroxide-dependent oxidation. We suggest that mixed disulfide formation by thiol transferase activity may help protect protein thiols from irreversible oxidation by heme-activated hydroperoxides.

UR - http://www.scopus.com/inward/record.url?scp=0026773311&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0026773311&partnerID=8YFLogxK

U2 - 10.1016/0003-9861(92)90530-A

DO - 10.1016/0003-9861(92)90530-A

M3 - Article

C2 - 1316737

AN - SCOPUS:0026773311

VL - 295

SP - 369

EP - 378

JO - Archives of Biochemistry and Biophysics

JF - Archives of Biochemistry and Biophysics

SN - 0003-9861

IS - 2

ER -