Alternative fluorimetric-based method to detect and compare total S-nitrosothiols in plants

Autor(es)

Mioto, Paulo Tamaso ; Rodriguez-Ruiz, Marta ; Mot, Augustin Catalin ; Zuccarelli, Rafael ; Corpas, Francisco J. ; Freschi, Luciano ; Mercier, Helenice

• Tipo de documento: Artigo Científico
• Fonte: NITRIC OXIDE-BIOLOGY AND CHEMISTRY; v. 68, p. 7-13, AUG 1 2017.
• Citações Web of Science: 1

Resumo

Nitric oxide (NO) is an important signaling molecule occurring in virtually all organisms, whose mechanism of action relies mainly on its interaction with proteins or peptides by nitrosylation, forming compounds such as S-nitrosothiols (SNO). The Saville reaction and the ozone-based chemiluminescence method are the main techniques used for nitrosylated protein quantification. While the Saville assay is not very sensitive, the ozone-based chemiluminescence is expensive and time-consuming. Here we propose a method in which the protein-bound NO groups are exposed to UV light, cleaving the bond and allowing the quantification of the derived NO molecules by diamino-rhodamine (DAR) dyes. The DAR based method was shown to be specific in plant tissues by pre-treatment of the samples with reducing agents and parallel EPR analysis. Spike-and-recovery assays revealed 72% recovery after a GSNO spike. Moreover, the method was significantly more sensitive than the Saville reaction, and this increase in sensitivity was crucial for detecting the reduced levels of nitrosylated proteins in plant species other than Arabidopsis. The method presented here is a suitable alternative to compare plant samples, allowing simple and fast detection of nitrosylated proteins. (C) 2017 Elsevier Inc. All rights reserved. (AU)

• Processo FAPESP: 13/18056-2 - Interação entre a sinalização luminosa, hormonal e do óxido nítrico durante a biogênese e diferenciação plastidial e acúmulo de compostos nutracêuticos em frutos de tomateiro
• Beneficiário: Luciano Freschi
• Modalidade de apoio: Auxílio à Pesquisa - Regular
• Processo FAPESP: 13/15108-1 - Biossíntese, transporte e modo de ação do óxido nítrico durante a indução do Metabolismo Ácido Crassuláceo (CAM) em Guzmania monostachia.
• Beneficiário: Paulo Tamaso Mioto
• Modalidade de apoio: Bolsas no Brasil - Doutorado
• Processo FAPESP: 11/50637-0 - Competência para a expressão do metabolismo ácido das crassuláceas (CAM) em bromélia epífita: sinalização, modulação da expressão, perfil transcricional e interação com o metabolismo de nitrogênio
• Beneficiário: Helenice Mercier
• Modalidade de apoio: Auxílio à Pesquisa - Temático