Metalloprotein-dependent decomposition of S-nitrosothiols: Studies on the stabilization and measurement of S-nitrosothiols in tissues

Ali R. Mani, Mohammad Ebrahimkhani, Silvia Ippolito, Richard Ollosson, Kevin P. Moore

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

The stabilization of S-nitrosothiols is critical for the development of assays to measure their concentration in tissues. Low-molecular-weight S-nitrosothiols are unstable in tissue homogenates, even in the presence of thiol blockers or metal-ion chelators. The aim of this study was to try and stabilize low-molecular-weight S-nitrosothiols in tissue and gain insight into the mechanisms leading to their decomposition. Rat tissues (liver, kidney, heart, and brain) were perfused and homogenized in the presence of a thiol-blocking agent (N-ethylmaleimide) and a metal-ion chelator (DTPA). Incubation of liver homogenate with low-molecular-weight S-nitrosothiols (l-CysNO, d-CysNO, and GSNO) resulted in their rapid decomposition in a temperature-dependent manner as measured by chemiluminescence. The decomposition of l-CysNO requires a cytoplasmic factor, with activity greatest in liver > kidney > heart > brain > plasma, and is inhibitable by enzymatic proteolysis or heating to 80°C, suggesting that a protein catalyzes the decomposition of S-nitrosothiols. The ability of liver homogenate to catalyze the decomposition of l-CysNO is up-regulated during endotoxemia and is dependent on oxygen, with the major product being nitrate. Multiple agents were tested for their ability to block the decomposition of l-CysNO without success, with the exception of potassium ferricyanide, which completely blocked CysNO decomposition in liver homogenates. This suggests that a ferrous protein (or group of ferrous proteins) may be involved. We also show that homogenization of tissues in ferricyanide-containing buffers in the presence of N-ethylmaleimide and DTPA can stabilize both low- and high-molecular-weight S-nitrosothiols in tissues before the measurement of their concentration.

Original languageEnglish (US)
Pages (from-to)1654-1663
Number of pages10
JournalFree Radical Biology and Medicine
Volume40
Issue number9
DOIs
StatePublished - May 1 2006
Externally publishedYes

Fingerprint

S-Nitrosothiols
Metalloproteins
Stabilization
Tissue
Liver
Decomposition
Molecular Weight
Molecular weight
Pentetic Acid
Ethylmaleimide
Chelating Agents
Sulfhydryl Compounds
Metal ions
Metals
Brain
Ions
Kidney
Proteins
Endotoxemia
Proteolysis

Keywords

  • Free radical
  • Iron-nitrosyl
  • N-nitrosamine
  • Nitric oxide
  • Rat
  • S-nitrosothiol
  • Thiol
  • Tissue

ASJC Scopus subject areas

  • Medicine(all)
  • Toxicology
  • Clinical Biochemistry

Cite this

Metalloprotein-dependent decomposition of S-nitrosothiols : Studies on the stabilization and measurement of S-nitrosothiols in tissues. / Mani, Ali R.; Ebrahimkhani, Mohammad; Ippolito, Silvia; Ollosson, Richard; Moore, Kevin P.

In: Free Radical Biology and Medicine, Vol. 40, No. 9, 01.05.2006, p. 1654-1663.

Research output: Contribution to journalArticle

@article{c7934ac3c1884f68b3a2254fd96ea8cd,
title = "Metalloprotein-dependent decomposition of S-nitrosothiols: Studies on the stabilization and measurement of S-nitrosothiols in tissues",
abstract = "The stabilization of S-nitrosothiols is critical for the development of assays to measure their concentration in tissues. Low-molecular-weight S-nitrosothiols are unstable in tissue homogenates, even in the presence of thiol blockers or metal-ion chelators. The aim of this study was to try and stabilize low-molecular-weight S-nitrosothiols in tissue and gain insight into the mechanisms leading to their decomposition. Rat tissues (liver, kidney, heart, and brain) were perfused and homogenized in the presence of a thiol-blocking agent (N-ethylmaleimide) and a metal-ion chelator (DTPA). Incubation of liver homogenate with low-molecular-weight S-nitrosothiols (l-CysNO, d-CysNO, and GSNO) resulted in their rapid decomposition in a temperature-dependent manner as measured by chemiluminescence. The decomposition of l-CysNO requires a cytoplasmic factor, with activity greatest in liver > kidney > heart > brain > plasma, and is inhibitable by enzymatic proteolysis or heating to 80°C, suggesting that a protein catalyzes the decomposition of S-nitrosothiols. The ability of liver homogenate to catalyze the decomposition of l-CysNO is up-regulated during endotoxemia and is dependent on oxygen, with the major product being nitrate. Multiple agents were tested for their ability to block the decomposition of l-CysNO without success, with the exception of potassium ferricyanide, which completely blocked CysNO decomposition in liver homogenates. This suggests that a ferrous protein (or group of ferrous proteins) may be involved. We also show that homogenization of tissues in ferricyanide-containing buffers in the presence of N-ethylmaleimide and DTPA can stabilize both low- and high-molecular-weight S-nitrosothiols in tissues before the measurement of their concentration.",
keywords = "Free radical, Iron-nitrosyl, N-nitrosamine, Nitric oxide, Rat, S-nitrosothiol, Thiol, Tissue",
author = "Mani, {Ali R.} and Mohammad Ebrahimkhani and Silvia Ippolito and Richard Ollosson and Moore, {Kevin P.}",
year = "2006",
month = "5",
day = "1",
doi = "10.1016/j.freeradbiomed.2006.01.004",
language = "English (US)",
volume = "40",
pages = "1654--1663",
journal = "Free Radical Biology and Medicine",
issn = "0891-5849",
publisher = "Elsevier Inc.",
number = "9",

}

TY - JOUR

T1 - Metalloprotein-dependent decomposition of S-nitrosothiols

T2 - Studies on the stabilization and measurement of S-nitrosothiols in tissues

AU - Mani, Ali R.

AU - Ebrahimkhani, Mohammad

AU - Ippolito, Silvia

AU - Ollosson, Richard

AU - Moore, Kevin P.

PY - 2006/5/1

Y1 - 2006/5/1

N2 - The stabilization of S-nitrosothiols is critical for the development of assays to measure their concentration in tissues. Low-molecular-weight S-nitrosothiols are unstable in tissue homogenates, even in the presence of thiol blockers or metal-ion chelators. The aim of this study was to try and stabilize low-molecular-weight S-nitrosothiols in tissue and gain insight into the mechanisms leading to their decomposition. Rat tissues (liver, kidney, heart, and brain) were perfused and homogenized in the presence of a thiol-blocking agent (N-ethylmaleimide) and a metal-ion chelator (DTPA). Incubation of liver homogenate with low-molecular-weight S-nitrosothiols (l-CysNO, d-CysNO, and GSNO) resulted in their rapid decomposition in a temperature-dependent manner as measured by chemiluminescence. The decomposition of l-CysNO requires a cytoplasmic factor, with activity greatest in liver > kidney > heart > brain > plasma, and is inhibitable by enzymatic proteolysis or heating to 80°C, suggesting that a protein catalyzes the decomposition of S-nitrosothiols. The ability of liver homogenate to catalyze the decomposition of l-CysNO is up-regulated during endotoxemia and is dependent on oxygen, with the major product being nitrate. Multiple agents were tested for their ability to block the decomposition of l-CysNO without success, with the exception of potassium ferricyanide, which completely blocked CysNO decomposition in liver homogenates. This suggests that a ferrous protein (or group of ferrous proteins) may be involved. We also show that homogenization of tissues in ferricyanide-containing buffers in the presence of N-ethylmaleimide and DTPA can stabilize both low- and high-molecular-weight S-nitrosothiols in tissues before the measurement of their concentration.

AB - The stabilization of S-nitrosothiols is critical for the development of assays to measure their concentration in tissues. Low-molecular-weight S-nitrosothiols are unstable in tissue homogenates, even in the presence of thiol blockers or metal-ion chelators. The aim of this study was to try and stabilize low-molecular-weight S-nitrosothiols in tissue and gain insight into the mechanisms leading to their decomposition. Rat tissues (liver, kidney, heart, and brain) were perfused and homogenized in the presence of a thiol-blocking agent (N-ethylmaleimide) and a metal-ion chelator (DTPA). Incubation of liver homogenate with low-molecular-weight S-nitrosothiols (l-CysNO, d-CysNO, and GSNO) resulted in their rapid decomposition in a temperature-dependent manner as measured by chemiluminescence. The decomposition of l-CysNO requires a cytoplasmic factor, with activity greatest in liver > kidney > heart > brain > plasma, and is inhibitable by enzymatic proteolysis or heating to 80°C, suggesting that a protein catalyzes the decomposition of S-nitrosothiols. The ability of liver homogenate to catalyze the decomposition of l-CysNO is up-regulated during endotoxemia and is dependent on oxygen, with the major product being nitrate. Multiple agents were tested for their ability to block the decomposition of l-CysNO without success, with the exception of potassium ferricyanide, which completely blocked CysNO decomposition in liver homogenates. This suggests that a ferrous protein (or group of ferrous proteins) may be involved. We also show that homogenization of tissues in ferricyanide-containing buffers in the presence of N-ethylmaleimide and DTPA can stabilize both low- and high-molecular-weight S-nitrosothiols in tissues before the measurement of their concentration.

KW - Free radical

KW - Iron-nitrosyl

KW - N-nitrosamine

KW - Nitric oxide

KW - Rat

KW - S-nitrosothiol

KW - Thiol

KW - Tissue

UR - http://www.scopus.com/inward/record.url?scp=33646050895&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=33646050895&partnerID=8YFLogxK

U2 - 10.1016/j.freeradbiomed.2006.01.004

DO - 10.1016/j.freeradbiomed.2006.01.004

M3 - Article

C2 - 16632125

AN - SCOPUS:33646050895

VL - 40

SP - 1654

EP - 1663

JO - Free Radical Biology and Medicine

JF - Free Radical Biology and Medicine

SN - 0891-5849

IS - 9

ER -