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

Abstract

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 α subunit of 17.5 kDa (α (17.5)) and two β subunits of 21.3 kDa and 22.6 kDa (β (21.3) and β (22.6)). The analysis from denaturing isoelectric focusing and two-dimension PAGE demonstrated that α (17.5), β (21.3) and β (22.6) had their pIs of 6.4, 5.3 and 5.4, respectively. Furthermore, mass spectroscopy analysis of β (21.3) and β (22.6) by MALDI-TOF mass spectrometry demonstrated the two β subunits had differences in peptide mass fingerprinting. These results revealed that the prepared R-phycocyanins were composed of one α and two β subunits. and , 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.