title: Alternative fluorimetric-based method to detect and compare total S-nitrosothiols in plants.
abstract: 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.
authors.affiliation: Department of Botany, Biological Sciences Center, Universidade Federal de Santa Catarina, Campus Reitor João David Ferreira Lima, s/n, 88040-900, Florianópolis, Brazil. Electronic address: paumioto@hotmail.com.
authors.forename: Paulo Tamaso
authors.initials: PT
authors.lastname: Mioto
authors.affiliation: Department of Biochemistry, Cell and Molecular Biology of Plants, Estación Experimental del Zaidín, CSIC, C/Profesor Albareda 1, E-18008, Granada, Spain.
authors.forename: Marta
authors.initials: M
authors.lastname: Rodríguez-Ruiz
authors.affiliation: Faculty of Chemistry and Chemical Engineering, Babeş-Bolyai University, 1 Mihail Kogălniceanu, 400084, Cluj Napoca, Romania.
authors.forename: Augustin Catalin
authors.initials: AC
authors.lastname: Mot
authors.affiliation: Department of Botany, Institute of Biosciences, Universidade de São Paulo, Rua do Matão 277, 05508-090, São Paulo, Brazil.
authors.forename: Rafael
authors.initials: R
authors.lastname: Zuccarelli
authors.affiliation: Department of Biochemistry, Cell and Molecular Biology of Plants, Estación Experimental del Zaidín, CSIC, C/Profesor Albareda 1, E-18008, Granada, Spain.
authors.forename: Francisco J
authors.initials: FJ
authors.lastname: Corpas
authors.affiliation: Department of Botany, Institute of Biosciences, Universidade de São Paulo, Rua do Matão 277, 05508-090, São Paulo, Brazil.
authors.forename: Luciano
authors.initials: L
authors.lastname: Freschi
authors.affiliation: Department of Botany, Institute of Biosciences, Universidade de São Paulo, Rua do Matão 277, 05508-090, São Paulo, Brazil.
authors.forename: Helenice
authors.initials: H
authors.lastname: Mercier
mesh_terms: D002623:Chemistry Techniques, Analytical / Q000295:instrumentation / Q000379:methods*; D003959:Diamines / Q000737:chemistry; D005470:Fluorometry; D057230:Limit of Detection; D010940:Plant Proteins / Q000032:analysis* / Q000737:chemistry; D010944:Plants / Q000737:chemistry*; D015203:Reproducibility of Results; D012235:Rhodamines / Q000737:chemistry; D026403:S-Nitrosothiols / Q000032:analysis* / Q000737:chemistry; D014466:Ultraviolet Rays
chemical_list: D003959:Diamines; D010940:Plant Proteins; D012235:Rhodamines; D026403:S-Nitrosothiols
keywords: Nitric oxide; Nitrosylated proteins; Ozone-based chemiluminescence; S-nitrosoglutathione; S-nitrosothiols; Saville