Metalloprotein-dependent decomposition of S-nitrosothiols: studies on the stabilization and measurement of S-nitrosothiols in tissues.
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 degrees 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.
Mani
Ali R
AR
The UCL Institute of Hepatology, Department of Medicine, Royal Free and University College Medical School, University College London, London NW3 2PF, UK.
Ebrahimkhani
Mohammad R
MR
Ippolito
Silvia
S
Ollosson
Richard
R
Moore
Kevin P
KP
D000818:Animals; D002614:Chelating Agents; D005292:Ferricyanides / Q000378:metabolism*; D008099:Liver / Q000737:chemistry / Q000378:metabolism*; D008297:Male; D008667:Metalloproteins / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D026403:S-Nitrosothiols / Q000032:analysis* / Q000378:metabolism*
D002614:Chelating Agents; D005292:Ferricyanides; D008667:Metalloproteins; D026403:S-Nitrosothiols; C028033:potassium ferricyanide