Purification and characterization of selenium-containing phycocyanin from selenium-enriched Spirulina platensis.
A fast protein liquid chromatographic method for purification of selenium-containing phycocyanin (Se-PC) from selenium-enriched Spirulina platensis was described in this study. The purification procedures involved fractionation by ammonium sulfate precipitation, DEAE-Sepharose ion-exchange chromatography and Sephacry S-300 size exclusion chromatography. The purity ratio (A620/A280) and the separation factor (A620/A655) of the purified Se-PC were 5.12 and 7.92, respectively. The Se concentration of purified Se-PC was 496.5 microg g(-1) protein, as determined by ICP-AES analysis. The purity of the Se-PC was further characterized by UV-VIS and fluorescence spectrometry, SDS-PAGE, RP-HPLC and gel filtration HPLC. The apparent molecular mass of the native Se-PC determined by gel filtration HPLC was 109 kDa, indicating that the protein existed as a trimer. SDS-PAGE of the purified Se-PC yielded two major bands corresponding to the alpha and beta subunits. A better separation of these two subunits was obtained by RP-HPLC. Identification of the alpha and beta subunits separated by SDS-PAGE and RP-HPLC was achieved by peptide mass fingerprinting (PMF) using MALDI-TOF-TOF mass spectrometry.
Chen
Tianfeng
T
Research Laboratory for Food Protein Production, Department of Biology, The Chinese University of Hong Kong, Hong Kong.
Wong
Yum-Shing
YS
Zheng
Wenjie
W
D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D008970:Molecular Weight; D010798:Phycocyanin / Q000737:chemistry* / Q000302:isolation & purification*; D021122:Protein Subunits / Q000302:isolation & purification / Q000378:metabolism; D012643:Selenium / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D013057:Spectrum Analysis; D053545:Spirulina / Q000737:chemistry* / Q000378:metabolism
D021122:Protein Subunits; D010798:Phycocyanin; D012643:Selenium