Unlike the formation of nitrosothiols by nitrous acid, our study revealed that NO2- effectively reacted with L-cysteine or reduced glutathione (GSH) at pH 7.0 and 7.4, to form orange-pink products of S-nitrosocysteine (CySNO) or S-nitrosoglutathione (GSNO). The reactions were in a concentration-dependent manner. These products exhibited not only peak absorbances at around 340 and 540 nm, but also unique colors and patterns of mobility on cellulose thin layer chromatographic plates. In comparison, the S-nitrosation of dithiothreitol was noted exclusively under acidic pH. In addition, the S-nitrosation of hemoglobin (Hb) by either peroxynitrite (PN) or NO2- at pH 6.0 was detected via Western blot. The half-life of degradation of CySNO in NO2- solution was significantly shorter than that of GSNO at a wide range of pH. In the absence of NO2-, degradation of GSNO was facilitated by incubation with L-cysteine, but not L-serine. In the signaling process involving NO -->PN --> NO2- --> CySNO/GSNO --> NO, L-cysteine may function as a NO-carrier to reach shorter-distance targets, and also an "activator" to release NO from GSNO. Furthermore, L-cysteine may play a vital role in reducing (severe) oxidative stress.
Peroxynitrite (PN)-pretreated histone III-S (NH) and reduced glutathione (GSH)-treated NH (NH(GSH)) were incubated with glutathione-S-transferase (GST) and glutathione peroxidase (GPX). Western blot analysis revealed decreased 3-nitrotyrosine immunoreactivity for NH(GSH), but not for NH. Additionally, increased nitrate was noted as an end product of these enzymatic reactions. The findings imply that GSH-treatment of NH may facilitate its conformational change in favor of subsequent enzymatic denitration and/or modification, which could be vital in relieving cellular oxidative stress and regulating NO/PN-mediated signal transduction cascade.
At neutral pH, S-nitrosoglutathione was formed by the reaction of reduced glutathione and sodium nitrite. The degradation of S-nitrosoglutathione, presumably by transnitrosation/denitrosation, was catalyzed by L-cysteine, or CoA-SH. Additionally, from the crude extract of rat brain, one protein with a large molecular mass was nitrosolated with nitrite, and was split into duplet peptides noted in Western blot. Furthermore, the incubation of nitrite with IgG may generate the intermediates of active nitrogen/oxygen species and lead to significant production of gas bubbles.
In various peroxynitrite (PN)-treated proteins, the formations of stable 3-nitrotyrosine (nitration) and labile S-nitrosocysteine (S-nitrosation) were observed by employing rapid Western blot in 6 h. The steps of SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and membrane-blotting were performed at 4 degrees C. It was noted that the intensity of immunoreactive bands specific for anti-nitrotyrosine was stronger than that specific for anti-S-nitrosocysteine. Additionally, the intensity was in the manner of a dose-dependency of PN. Nitration/S-nitrosation were formed in the following treated proteins, including bovine serum albumin (BSA), DNase-1, ceruloplasmin, catalase and hemoglobin (Hb). The incubation of PN-pretreated hemoglobin with 1 mM reduced glutathione (GSH) did not change immunoreactivity significantly. However, the addition of glutathione S-transferase (GST) or glutathione peroxidase (GPX) to the above incubation mixture, resulted in decreased immunoreactivity, suggesting GSH may form a transition complex with PN-pretreated hemoglobin and/or partially reduce/modify the treated hemoglobin, thereby increasing the accessibility for the subsequent modification by GST or GPX. Such decreased immunoreactivity indicates that nitrotyrosine and S-nitrosocysteine of treated hemoglobin was, indeed, further modified via (a) converting -NO2 to -NH2 in tyrosine residues, (b) denitrating -NO2 directly/indirectly in tyrosine residues, and/or (c) changing -S-NO to -SH in cysteine residues, or denitrosation. The findings imply similar enzymatic modifications of proteins may also occur in vivo, and therefore play a pivotal role in the NO-related cellular signaling cascade(s).
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.