Formation and detection of oxidant-generated tryptophan dimers in peptides and proteins

Luke Carroll; David I Pattison; Justin B Davies; Robert F Anderson; Camilo Lopez-Alarcon; Michael J Davies

doi:10.1016/j.freeradbiomed.2017.09.020

Formation and detection of oxidant-generated tryptophan dimers in peptides and proteins

Luke Carroll, David I Pattison, Justin B Davies, Robert F Anderson, Camilo Lopez-Alarcon, Michael J Davies

Inflammation, Metabolism and Oxidation

32 Citations (Scopus)

Abstract

Free radicals are produced during physiological processes including metabolism and the immune response, as well as on exposure to multiple external stimuli. Many radicals react rapidly with proteins resulting in side-chain modification, backbone fragmentation, aggregation, and changes in structure and function. Due to its low oxidation potential, the indole ring of tryptophan (Trp) is a major target, with this resulting in the formation of indolyl radicals (Trp•). These undergo multiple reactions including ring opening and dimerization which can result in protein aggregation. The factors that govern Trp• dimerization, the rate constants for these reactions and the exact nature of the products are not fully elucidated. In this study, second-order rate constants were determined for Trp• dimerization in Trp-containing peptides to be 2-6 × 108M-1s-1 by pulse radiolysis. Peptide charge and molecular mass correlated negatively with these rate constants. Exposure of Trp-containing peptides to steady-state radiolysis in the presence of NaN3 resulted in consumption of the parent peptide, and detection by LC-MS of up to 4 different isomeric Trp-Trp cross-links. Similar species were detected with other oxidants, including CO3•- (from the HCO3- -dependent peroxidase activity of bovine superoxide dismutase) and peroxynitrous acid (ONOOH) in the presence or absence of HCO3-. Trp-Trp species were also isolated and detected after alkaline hydrolysis of the oxidized peptides and proteins. These studies demonstrate that Trp• formed on peptides and proteins undergo rapid recombination reactions to form Trp-Trp cross-linked species. These products may serve as markers of radical-mediated protein damage, and represent an additional pathway to protein aggregation in cellular dysfunction and disease.

Original language	English
Journal	Free Radical Biology & Medicine
Volume	113
Pages (from-to)	132-142
Number of pages	11
ISSN	0891-5849
DOIs	https://doi.org/10.1016/j.freeradbiomed.2017.09.020
Publication status	Published - Dec 2017

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10.1016/j.freeradbiomed.2017.09.020

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title = "Formation and detection of oxidant-generated tryptophan dimers in peptides and proteins",

abstract = "Free radicals are produced during physiological processes including metabolism and the immune response, as well as on exposure to multiple external stimuli. Many radicals react rapidly with proteins resulting in side-chain modification, backbone fragmentation, aggregation, and changes in structure and function. Due to its low oxidation potential, the indole ring of tryptophan (Trp) is a major target, with this resulting in the formation of indolyl radicals (Trp•). These undergo multiple reactions including ring opening and dimerization which can result in protein aggregation. The factors that govern Trp• dimerization, the rate constants for these reactions and the exact nature of the products are not fully elucidated. In this study, second-order rate constants were determined for Trp• dimerization in Trp-containing peptides to be 2-6 × 108M-1s-1 by pulse radiolysis. Peptide charge and molecular mass correlated negatively with these rate constants. Exposure of Trp-containing peptides to steady-state radiolysis in the presence of NaN3 resulted in consumption of the parent peptide, and detection by LC-MS of up to 4 different isomeric Trp-Trp cross-links. Similar species were detected with other oxidants, including CO3•- (from the HCO3- -dependent peroxidase activity of bovine superoxide dismutase) and peroxynitrous acid (ONOOH) in the presence or absence of HCO3-. Trp-Trp species were also isolated and detected after alkaline hydrolysis of the oxidized peptides and proteins. These studies demonstrate that Trp• formed on peptides and proteins undergo rapid recombination reactions to form Trp-Trp cross-linked species. These products may serve as markers of radical-mediated protein damage, and represent an additional pathway to protein aggregation in cellular dysfunction and disease.",

author = "Luke Carroll and Pattison, {David I} and Davies, {Justin B} and Anderson, {Robert F} and Camilo Lopez-Alarcon and Davies, {Michael J}",

year = "2017",

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doi = "10.1016/j.freeradbiomed.2017.09.020",

language = "English",

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journal = "Free Radical Biology & Medicine",

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T1 - Formation and detection of oxidant-generated tryptophan dimers in peptides and proteins

AU - Carroll, Luke

AU - Pattison, David I

AU - Davies, Justin B

AU - Anderson, Robert F

AU - Lopez-Alarcon, Camilo

AU - Davies, Michael J

PY - 2017/12

Y1 - 2017/12

N2 - Free radicals are produced during physiological processes including metabolism and the immune response, as well as on exposure to multiple external stimuli. Many radicals react rapidly with proteins resulting in side-chain modification, backbone fragmentation, aggregation, and changes in structure and function. Due to its low oxidation potential, the indole ring of tryptophan (Trp) is a major target, with this resulting in the formation of indolyl radicals (Trp•). These undergo multiple reactions including ring opening and dimerization which can result in protein aggregation. The factors that govern Trp• dimerization, the rate constants for these reactions and the exact nature of the products are not fully elucidated. In this study, second-order rate constants were determined for Trp• dimerization in Trp-containing peptides to be 2-6 × 108M-1s-1 by pulse radiolysis. Peptide charge and molecular mass correlated negatively with these rate constants. Exposure of Trp-containing peptides to steady-state radiolysis in the presence of NaN3 resulted in consumption of the parent peptide, and detection by LC-MS of up to 4 different isomeric Trp-Trp cross-links. Similar species were detected with other oxidants, including CO3•- (from the HCO3- -dependent peroxidase activity of bovine superoxide dismutase) and peroxynitrous acid (ONOOH) in the presence or absence of HCO3-. Trp-Trp species were also isolated and detected after alkaline hydrolysis of the oxidized peptides and proteins. These studies demonstrate that Trp• formed on peptides and proteins undergo rapid recombination reactions to form Trp-Trp cross-linked species. These products may serve as markers of radical-mediated protein damage, and represent an additional pathway to protein aggregation in cellular dysfunction and disease.

AB - Free radicals are produced during physiological processes including metabolism and the immune response, as well as on exposure to multiple external stimuli. Many radicals react rapidly with proteins resulting in side-chain modification, backbone fragmentation, aggregation, and changes in structure and function. Due to its low oxidation potential, the indole ring of tryptophan (Trp) is a major target, with this resulting in the formation of indolyl radicals (Trp•). These undergo multiple reactions including ring opening and dimerization which can result in protein aggregation. The factors that govern Trp• dimerization, the rate constants for these reactions and the exact nature of the products are not fully elucidated. In this study, second-order rate constants were determined for Trp• dimerization in Trp-containing peptides to be 2-6 × 108M-1s-1 by pulse radiolysis. Peptide charge and molecular mass correlated negatively with these rate constants. Exposure of Trp-containing peptides to steady-state radiolysis in the presence of NaN3 resulted in consumption of the parent peptide, and detection by LC-MS of up to 4 different isomeric Trp-Trp cross-links. Similar species were detected with other oxidants, including CO3•- (from the HCO3- -dependent peroxidase activity of bovine superoxide dismutase) and peroxynitrous acid (ONOOH) in the presence or absence of HCO3-. Trp-Trp species were also isolated and detected after alkaline hydrolysis of the oxidized peptides and proteins. These studies demonstrate that Trp• formed on peptides and proteins undergo rapid recombination reactions to form Trp-Trp cross-linked species. These products may serve as markers of radical-mediated protein damage, and represent an additional pathway to protein aggregation in cellular dysfunction and disease.

U2 - 10.1016/j.freeradbiomed.2017.09.020

DO - 10.1016/j.freeradbiomed.2017.09.020

M3 - Journal article

C2 - 28962874

SN - 0891-5849

VL - 113

SP - 132

EP - 142

JO - Free Radical Biology & Medicine

JF - Free Radical Biology & Medicine

ER -

Formation and detection of oxidant-generated tryptophan dimers in peptides and proteins

Abstract

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