Isolation, Purification, Polypeptide Composition Analysis Of The Four R-phycoerythrins From A Marine Red Alga Polysiphonia Urceolata Grev

Phycobiliproteins from a marine red macroalga Polysiphonia urceolata Grev were extracted in 50 mmol/L phosphate buffer (pH=7.0) when the alga was disrupted by ultrasonication, and then they were precipitated by salting-out with ammonium sulfate. Four R-phycoerythrins (R-PEs) and an R-phycocyanin (R-PC) with different electric charges were isolated from the phycobiliprotein extract by the ion exchange chromatography on DEAE-52, and the four isolated R-phycoerythrins, named PE₁₆₉-1,PE₂₆₇-1, PE₃₅₆-2 and PE₅₂₃-2, were the objects of this study. Then the four R-phycoerythrins were purified by the gel filtration chromatography on Sephadex G-150 and Sephacryl S-300. The four prepared R-PEs have very similar spectral properties: they showed three absorption peaks at about 498 nm, 540 nm and 565 nm, and gave a fluorescent emission peak at about 576 nm. PE₂₆₇-1, PE₃₅₆-2 and PE₅₂₃-2, which were different from PE₁₆₉-1, showed a single band both in native-PAGE and in native isoelectric focusing (IEF). Isoelectric points (pIs) of PE₂₆₇-1, PE₃₅₆-2 and PE₅₂₃-2 are 4.5, 4.4 and 4.4, respectively. PE₁₆₉-1 had a low content in the phycobiliprotein extract. The analysis from native-PAGE and IEF, denaturing-IEF and SDS-PAGE demonstrated that PE₁₆₉-1 was probably some low molecular mass PEs originated from the dissociation of hexamer phycoerythrins.Polypeptide composition analysis of the four prepared R-PEs by SDS-PAGE, denaturing-IEF and two-dimension PAGE (2D-PAGE) demonstrated that PE₂₆₇-1,PE₃₅₆-2 and PE₅₂₃-2 contained the same chromorphe-carring colored subunits, and the masses of these subunits were 17.5 kDa (α), 19.5 kDa (β) and 34.4 kDa (γ), respectively. The three subunits had proportions of 3α:3β:1γ. Compared with PE₂₆₇-1, both of PE₃₅₆-2 and PE₅₂₃-2 have a colorless polypeptide of 45.7 kDa (Lnk45.7). Besides Lnk45.7, PE₅₂₃-2 also had one more colorless polypeptide of 62.1 kDa (Lnk^62.1). The three subunits and the two colorless polypeptide showed proportions of 6α:6β:2γ:1Lnk45.7, and 6α:6β:2γ:0.5Lnk^62.1. The colorless polypeptides Lnk45.7 and Lnk^62.1, like subunitγ, probably function as the linkage polypeptides to connect hexamer R-PEs in rod domains. These results indicate that PE₂₆₇-1, PE₃₅₆-2 and PE₅₂₃-2 mainly exist in different forms of hexamer complexes, (αβ)3-γ-(αβ)3-γ,γ-(αβ)3-γ-(αβ)3-Lnk45.7 andγ-(αβ)3-γ-(αβ)3-Lnk^62.1. In the 2D-PAGE, theαsubunit showed three different forms with pIs at 5.95, 5.80 and 5.65; and theβsubunit had five different forms, pIs of which were 5.56, 5.46, 5.33, 5.25 and 5.11, respectively. The results from this work gave worthful data to the investigation on R-PE hexamers and assembly of rod domains in phycobilisomes of marine red macroalgae. The established procedures of chromatography, PAGE, IEF and 2D-PAGE here can also be used in the investigation on phycobiliprotein composition of other red macroalgae.