Isolation, Purification and Properties of an R-Phycocyanin from the Phycobilisomes of a Marine Red Macroalga Polysiphonia urceolata

Phycobilisomes were prepared from a marine red macroalga Polysiphonia urceolata (P. urceolata) by sucrose step-gradient ultracentrifugation. From the prepared phycobilisomes, an R-phycocyanin was isolated by gel filtration on Sephadex G-150 and then purified by ion exchange chromatography on DEAE-Sepharose Fast Flow and native polyacrylamide gel electrophoresis (PAGE) performed in neutral buffer systems. The purified R-phycocyanins showed not only a homogeneous trimer of 136 kDa in gel filtration and a single band in native PAGE, but also exhibited one band at about pH 5.7 in native isoelectric focusing (IEF). By a gradient SDS-PAGE the purified R-phycocyanin was determined to contain one alpha subunit of 17.5 kDa (alpha(17.5)) and two beta subunits of 21.3 kDa and 22.6 kDa (beta(21.3) and beta(22.6)). The analysis from denaturing isoelectric focusing and two-dimension PAGE demonstrated that alpha(17.5), beta(21.3) and beta(22.6) had their pIs of 6.4, 5.3 and 5.4, respectively. Furthermore, mass spectroscopy analysis of beta(21.3) and beta(22.6) by MALDI-TOF mass spectrometry demonstrated the two beta subunits had differences in peptide mass fingerprinting. These results revealed that the prepared R-phycocyanins were composed of one alpha and two beta subunits. (alpha(17.5)(3) beta(21.3)(2) beta(22.6)(1)) and (alpha(17.5)(3) beta(21.3)(2) beta(22.6)(1)), which have a structural foundation to show their pIs too close for them to be definitely resolved by native IEF, are postulated to be the most possible trimeric forms of the R-phycocyanins prepared from the phycobilisomes of P. urceolata.