Biochemical,physicochemical and molecular characterization of a genuine 2-Cys-peroxiredoxin purified from cowpea [Vigna unguiculata (L.) Walpers] leaves |
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Authors: | Fredy DA Silva Ilka M Vasconcelos Marina DP Lobo Patrícia G de Castro Vladimir G Magalhães Cléverson DT de Freitas Célia RRS Carlini Paulo M Pinto Leila M Beltramini José HA Filho Eduardo B Barros Luciana MR Alencar Thalles B Grangeiro José TA Oliveira |
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Institution: | 1. Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará (UFC), CE, Brazil;2. Departamento de Biologia, Universidade Federal do Ceará (UFC), CE, Brazil;3. Departamento de Biologia, Universidade Federal do Piauí (UFPI), PI, Brazil;4. Departamento de Biofísica, Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul (UFRGS), RS, Brazil;5. Instituto de Física de São Carlos (IFSC), Universidade de São Paulo (USP), SP, Brazil;6. Departamento de Biologia, Universidade Regional do Rio Grande do Norte (UERN), RN, Brazil;g Departamento de Física, Universidade Federal do Ceará (UFC), CE, Brazil |
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Abstract: | BackgroundPeroxiredoxins 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).MethodsVu-2-Cys-Prx was purified from leaves by ammonium sulfate fractionation, chitin affinity and ion exchange chromatography.ResultsVu-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.General significanceThe 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. |
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Keywords: | BSA bovine serum albumin CS citrate synthase CBD chitin-binding domain 2-Cys-Prx 2-Cys-Peroxiredoxin Vu-2-Cys-Prx Vigna unguiculata 2-Cys-peroxiredoxin CD circular dichroism H2O2 hydrogen peroxide IPG buffer ampholyte-containing buffer concentrate IPG strips Immobiline&trade DryStrip gels for isoelectric focusing of proteins DTT Dithiothreitol ESI-Q-TOF MS/MS electrospray ionization quadrupole time-of-flight NADPH nicotinamide adenine dinucleotide phosphate |
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