[Purification of plastocynin from Ulva pertusa by column chromatography and analysis of its N-terminal amino acid sequence].
Protein plastocyanin from a green alga, Ulva pertusa, has been purified. Samples were homogenized in 0.02 mol/L phosphate buffer (pH 7.2) and then centrifuged to remove debris and subjected to ammonium sulfate fractionation (40%-80% saturation). Ion exchange column chromatography with DEAE-Sepharose Fast Flow and gel filtration column chromatography with Sephadex G-75 were then employed for further purification of plastocyanin. Three peaks, A, B and C, were eluted with 0.01 mol/L phosphate buffer, containing a NaCl linear gradient from 0 to 1.0 mol/L at the flow rate of 32 mL/h through DEAE-Sepharose chromatography. The protein fractions containing the plastocyanin were then purified further with Sephardex G-75 column chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophores (SDS-PAGE) is analysis indicates that the protein was purified to homogeneity and its relative molecular mass is 10,000. N-terminal amino acid sequence was used to identify the protein. The protein was transblotted to PVDF membrane and N-terminal amino acid sequence was performed via Edman degradation with an automated amino acid sequencer. The 20 N-terminal amino acid residues are AAIVKLGPDDGSLAFVPSKI, which share 85% homology with the 20 N-terminal amino acid sequence of U. prolifera and U. arasakii, and share 90% homology with the ones of U. pertusa formerly reported.
Liu
Zhenyu
Z
Institute of Plant Virology, Fujian Agriculture and Forestry University, Fuzhou, China. cosmosliu@163.com
Wu
Zujian
Z
Lin
Qiying
Q
Xie
Lianhui
L
D000460:Chlorophyta; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010970:Plastocyanin / Q000737:chemistry / Q000302:isolation & purification*; D020539:Sequence Analysis, Protein; D044445:Ulva / Q000737:chemistry*
D010970:Plastocyanin