Elucidating the structure of two cyclotides of Viola tianshanica maxim by MALDI TOF/TOF MS analysis.
- Author:
Bin XIANG
;
Guohua DU
;
Xuchen WANG
;
Shuxiang ZHANG
;
Xianyun QIN
;
Jianqiang KONG
;
Kedi CHENG
;
Yongji LI
;
Wei WANG
- Publication Type:Journal Article
- From:
Acta Pharmaceutica Sinica
2010;45(11):1402-9
- CountryChina
- Language:Chinese
-
Abstract:
The cyclotides are a family of cyclic "mini" proteins that occur in Violaceae, Rubiaceae and Cucurbitaceae plant families and contain a head-to-tail cyclic backbone and a cystine knot arranged by three disulfide bonds. To study the natural cyclotides of V tianshanica, dried herb was extracted with 50% ethanol, and the concentrated aqueous extract was subjected to a solvent-solvent partitioning between water and hexane, ethyl acetate and n-butanol, separately. The n-butanol extract containing cyclotides was subjected to column chromatography over Sephadex LH-20, eluted with 30% methanol. The subfractions were directly reduced by DTT and analyzed by reverse-phase HPLC. The peaks with different retention times were shown on the profile of RP-HPLC and collected. The cyclotides were speculated based on masses range from 3 000 to 3 500 Da. The purified cyclotides were reduced with DTT, alkylated with iodoacetamide, and then were cleaved with endoproteinase Glu-C, endoproteinase Lys-C and Trypsin, separately. The digested peptides were purified on RP-HPLC and analyzed on MALDI TOF/TOF analyzer. A new cyclotide, cycloviolacin T1 and a reported cyclotide varv E were systemically determined using MALDI TOF/TOF system. So the method for the isolation and characterization of cyclotides was quickly built up in succession.
