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

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• 作者：Fredy D.A. Silva^a ; Ilka M. Vasconcelos^a ; Marina D.P. Lobo^a ; Patrí ; cia G. de Castro^a ; Vladimir G. Magalhã ; es^a ; Clé ; verson D.T. de Freitas^c ; Cé ; lia R.R.S. Carlini^d ; Paulo M. Pinto^d ; Leila M. Beltramini^e ; José ; H.A. Filho^f ; Eduardo B. Barros^g ; Luciana M.R. Alencar^g ; Thalles B. Grangeiro^b ; ^{tbgrangeiro@gmail.com} ; José ; T.A. Oliveira^a ; ^{jtaolive@ufc.br}
• 关键词：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 conc
• 刊名：Biochimica et Biophysica Acta (BBA)/General Subjects
• 出版年：2012
• 期刊代码：16_03044165
• 类别：bio
• 出版时间：July, 2012
• 卷：1820
• 期：7
• 页码：1128-1140
• 文件大小：2151 K

摘要

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%伪-helix, 39%尾-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 significance

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.