Tetramethylbenzidine-H₂O₂ 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.