Biochemical, physicochemical and molecular characterization of a genuine 2-Cys-peroxiredoxin purified from cowpea [Vigna unguiculata (L.) Walpers] leaves

• Published in: Biochimica et biophysica acta Vol. 1820; no. 7; pp. 1128 - 1140
• Main Authors: Silva, Fredy D.A., Vasconcelos, Ilka M., Lobo, Marina D.P., de Castro, Patrícia G., Magalhães, Vladimir G., de Freitas, Cléverson D.T., Carlini, Célia R.R.S., Pinto, Paulo M., Beltramini, Leila M., Filho, José H.A., Barros, Eduardo B., Alencar, Luciana M.R., Grangeiro, Thalles B., Oliveira, José T.A.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.07.2012
• Subjects: 2-Cys-peroxiredoxin; Amino Acid Sequence; amino acids; ammonium sulfate; Antioxidants - metabolism; atomic force microscopy; cDNA sequence; chitin; Circular dichroism; citrate (si)-synthase; complementary DNA; cowpeas; Cysteine - chemistry; denaturation; disulfide bonds; Electrophoresis, Gel, Two-Dimensional; Electrophoresis, Polyacrylamide Gel; Fabaceae - growth & development; Fabaceae - metabolism; fractionation; gel chromatography; heat stability; hydrogen peroxide; Hydrogen Peroxide - metabolism; Hydrogen-Ion Concentration; insulin; ion exchange chromatography; leaves; Microscopy, Atomic Force; molecular chaperones; Molecular Chaperones - metabolism; Molecular Sequence Data; molecular weight; NADP (coenzyme); Oxidation-Reduction; Peroxiredoxins - isolation & purification; Peroxiredoxins - metabolism; Phaseolus vulgaris; Plant Leaves - growth & development; Plant Leaves - metabolism; plasmids; polyacrylamide gel electrophoresis; Populus; purification; Sequence Homology, Amino Acid; Tandem Mass Spectrometry; Vigna unguiculata; ESI-Q-TOF MS/MS; NADPH; cDNA sequence; CD; CBD; DTT; Vigna unguiculata; Amino acid sequence; Vu-2-Cys-Prx; IPG buffer; H2O2; 2-Cys-peroxiredoxin; Circular dichroism; CS; BSA; 2-Cys-Prx; IPG strips
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/j.bbagen.2012.03.003

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). Vu-2-Cys-Prx was purified from leaves by ammonium sulfate fractionation, chitin affinity and ion exchange chromatography. Vu-2-Cys-Prx reduces H2O2 using NADPH and DTT. Vu-2-Cys-Prx is a 44kDa (SDS-PAGE)/46kDa (exclusion chromatography) protein that appears as a 22kDa 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 28.622kDa/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. 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. ► A 2-Cys-Prx (Vu-2-Cys-Prx) was purified from cowpea (Vigna unguiculata) leaves. ► Vu-2-Cys-Prx binds to chitin, is a 44/46kDa protein with pI of 4.56–4.72. ► Its Cys52, Pro45, Thr49, and Arg128 residues are conserved as in all other 2-Cys-Prx. ► Vu-2-Cys-Prx prevented DNA degradation, forms decamers, and has chaperone activity.