Multi Siphonales Phycobilisomes R-phycoerythrin And R-phycocyanin Spectral And Structural Characteristics,

The phycobilisomes of a marine red macroalga Polysiphonia urceolata were extracted in 900 mM phosphate buffer (pH 7.0), then solublized with 2% (v/v) NP-40 and finaly prepared by ultracentrifugation on a step-gradient of sucrose concentration.By a process of combining Sepharose CL-4B/Sephadex G-150 chromatography sequentially with ion exchange chromatography and native PAGE, an R-phycoerythrin (R-PE) and an R-phycocyanin (R-PC) were isolated and purified fron the dissociated phycobilisomes. The prepared R-PE has three absorption peaks at 497 nm,538 nm and 566 nm, and it has a fluorescent emission peak at 576 nm. The prepared R-PC shows two absorption peaks at 549 nm and 617 nm, and it has a fluorescent emission peak at 636 nm. Both of the R-PE and R-PC showed single band in the native PAGE performed in a neutral buffer system. The molecular size analysis by gel filtration on Superdex-200 showed that the R-PE is 240 kDa and the R-PC is 136 kDa. These results demonstrate that the prepared R-PE is a hexamer and the prepared R-PC is a trimer. However, the prepared R-PE was resolved into two bands with close pI at pH 4.7 by the native IEF in a pH gradient from 4.0 to 6.5, and the prepared R-PC was also resolved into two bands with very close pI at pH 5.7. This reveals that the R-PE may exist in two different forms of hexamers and the R-PC may exist in two different forms of trimers. The two forms of hexamers/trimers showed homogenious molecular size in the Superdex-200 chromatogrphy and exhibited homogenious ratios of charge to mass in the native PAGE.Analysis of polypeptide composition by SDS-PAGE, denaturing IEF and two-dimension PAGE (2D-PAGE) demonstrated that the prepared R-PE is composed of four subunits,α17.3,β19.5,γ33.3 andγ'31.0, and it shows subunit proportionsα17.3:β19.5:γ33.3:γ31.0=6:6:2:1. Of the four subunits, theα17.3 has pI 5.0, theβ19.5 has pI 5.8 and the twoγofγ33.3 andγ'31.0 show pIs within 5.0-5.8. The prepared R-PC was proved to have three subunits,α17.5,β21.3 andβ'22.3 and pIs of them are 6.4,5.3 and 5.4, respectively. Based on the characteristics of the subunits of the two prepared phycobiliproteins, the most possible two forms of the trimeric R-PC are (α317.5β221.3β122.6) and (α317.5β121.3β222.6),which have the least differences in molecular size, in the ratio of charge to mass and in pI, and for the R-PE the most possible forms of hexamers areγ(αβ)3-γ-(αβ)3γ' and γ(αβ)3-γ'(αβ)3γ.The fact that each of the two hexamers contains at least twoγsubunits (twoγones or oneγand oneγ') reveals that when a trimeric R-PE (αβ)3 adds in the rod domain of the phycobilisomes, there is oneγsubunit which is employed to bind the trimer (αβ)3 to the rod domain, making the rod domain prolonged. The established procedures of chromatography, PAGE, IEF and 2D-PAGE in this work can provide a valuable reference for investigations on the phycobilisome and and its phycobiliprotein composition of other red macroalgae.