Biochemical, physicochemical and molecular characterization of a genuine 2-Cys-peroxiredoxin purified from cowpea [Vigna unguiculata (L.) Walpers] leaves.
BACKGROUND
Peroxiredoxins have diverse functions in cellular defense-signaling pathways. 2-Cys-peroxiredoxins (2-Cys-Prx) reduce H2O2 and alkyl-hydroperoxide. This study describes the purification and characterization of a genuine 2-Cys-Prx from Vigna unguiculata (Vu-2-Cys-Prx).


METHODS
Vu-2-Cys-Prx was purified from leaves by ammonium sulfate fractionation, chitin affinity and ion exchange chromatography.


RESULTS
Vu-2-Cys-Prx reduces H2O2 using NADPH and DTT. Vu-2-Cys-Prx is a 44 kDa (SDS-PAGE)/46 kDa (exclusion chromatography) protein that appears as a 22 kDa molecule under reducing conditions, indicating that it is a homodimer linked intermolecularly by disulfide bonds and has a pI range of 4.56–4.72; its NH2-terminal sequence was similar to 2-Cys-Prx from Phaseolus vulgaris (96%) and Populus tricocarpa (96%). Analysis by ESI-Q-TOF MS/MS showed a molecular mass/pI of 28.622 kDa/5.18. Vu-2-Cys-Prx has 8% α-helix, 39% β-sheet, 22% of turns and 31% of unordered forms. Vu-2-Cys-Prx was heat stable, has optimal activity at pH 7.0, and prevented plasmid DNA degradation. Atomic force microscopy shows that Vu-2-Cys-Prx oligomerized in decamers which might be associated with its molecular chaperone activity that prevented denaturation of insulin and citrate synthase. Its cDNA analysis showed that the redox-active Cys52 residue and the amino acids Pro45, Thr49 and Arg128 are conserved as in other 2-Cys-Prx.


CONCLUSIONS
The biochemical and molecular features of Vu-2-Cys-Prx are similar to other members of 2-Cys-Prx family. To date, only one publication reported on the purification of native 2-Cys-Prx from leaves and the subsequent analysis by N-terminal Edman sequencing, which is crucial for construction of stromal recombinant 2-Cys-Prx proteins.
Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará, CE, Brazil.
Fredy D A
FD
Silva
Ilka M
IM
Vasconcelos
Marina D P
MD
Lobo
Patrícia G
PG
de Castro
Vladimir G
VG
Magalhães
Cléverson D T
CD
de Freitas
Célia R R S
CR
Carlini
Paulo M
PM
Pinto
Leila M
LM
Beltramini
José H A
JH
Filho
Eduardo B
EB
Barros
Luciana M R
LM
Alencar
Thalles B
TB
Grangeiro
José T A
JT
Oliveira
D000595:Amino Acid Sequence; D000975:Antioxidants / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry*; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D007887:Fabaceae / Q000254:growth & development / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D018625:Microscopy, Atomic Force; D018832:Molecular Chaperones / Q000378:metabolism*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D054464:Peroxiredoxins / Q000302:isolation & purification* / Q000378:metabolism*; D018515:Plant Leaves / Q000254:growth & development / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D053719:Tandem Mass Spectrometry
D000975:Antioxidants; D018832:Molecular Chaperones; D006861:Hydrogen Peroxide; D054464:Peroxiredoxins; D003545:Cysteine