Identification of a 2-cys peroxiredoxin as a tetramethyl benzidine-hydrogen peroxide stained protein from the thylakoids of the extreme halophyte Arthrocnemum macrostachyum L.
Tetramethylbenzidine-H(2)O(2) staining of SDS-polyacrylamide gel is a widely used method for the specific detection of proteins with heme-dependent peroxidase activity. When this method was used with thylakoids from the halophytic plant Arthrocnemum macrostachyum, besides the cytochrome f and cytochrome b6 proteins usually found in higher plants and cyanobacteria, at least four additional bands were detected. One of them, a 46-kDa protein, was shown to be an extrinsic protein, and identified by mass spectrometry and immunoblotting as a 2-cys peroxiredoxin. Peroxidase activity was insensitive to oxidizing agents such as trans-4,4-diydroxy-1,2-dithiane or hydrogen peroxide, but was inhibited by treatment of thylakoids with reducing agents such as dithiothreitol or mercaptoethanol. By immunoblotting, it was shown that loss of peroxidase activity was paralleled by disappearance of the 46-kDa band, which was converted to a 23-kDa immunoreactive form. A dimer/monomer relationship between the two proteins is suggested, with the dimeric form likely being a heme-binding protein. This possibility was further supported by anionic exchange chromatography and de novo sequencing of tryptic fragments of the protein and sequence comparison, as most of the residues previously implicated in heme binding in 2-cys peroxiredoxin from Rattus norvegicus were conserved in A. macrostachyum. The amount of this protein was modulated by environmental conditions, and increased when salt concentration in the growth medium was higher or lower than the optimal one.
Dipartimento di Scienze dell'Ambiente e della Vita, Università del Piemonte Orientale Amedeo Avogadro, Alessandria, Novara, Vercelli, Italy. andrea.trotta@utu.fi
Andrea
A
Trotta
Alessia
A
Antonacci
Francesco
F
Marsano
Susana
S
Redondo-Gomez
Enrique Manuel
EM
Figueroa Clemente
Flora
F
Andreucci
Roberto
R
Barbato
D027703:Amaranthaceae / Q000378:metabolism*; D001560:Benzidines / Q000737:chemistry*; D006861:Hydrogen Peroxide / Q000737:chemistry*; D054464:Peroxiredoxins / Q000737:chemistry / Q000378:metabolism*; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism*; D055051:Salt-Tolerant Plants / Q000378:metabolism*; D020524:Thylakoids / Q000378:metabolism*
D001560:Benzidines; D010940:Plant Proteins; C021758:3,3',5,5'-tetramethylbenzidine; D006861:Hydrogen Peroxide; D054464:Peroxiredoxins