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 language | English (US) |
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Pages (from-to) | 1654-1663 |
Number of pages | 10 |
Journal | Free Radical Biology and Medicine |
Volume | 40 |
Issue number | 9 |
DOIs | |
State | Published - May 1 2006 |
Keywords
- Free radical
- Iron-nitrosyl
- N-nitrosamine
- Nitric oxide
- Rat
- S-nitrosothiol
- Thiol
- Tissue
ASJC Scopus subject areas
- Biochemistry
- Physiology (medical)