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

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

Research output: Contribution to journalArticle

17 Scopus citations

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

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Keywords

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

ASJC Scopus subject areas

  • Biochemistry
  • Physiology (medical)

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