Isolation And Characterization Of Phycobiliprotein Of A Red Algae Polysiphonia Urceolata Grev

Polysiphonia urceolata is one of the marine red macro algae. Phycobilisomes of red algae are in the form of macromolecular complexes attached to the stromatic side of the thylakoid membrane, which harvest the sunlight energy and transfer it to photosystemⅡ(PSⅡ).At present, prokaryotic cyanobacteria and green plants have been the main objects of study on the light reaction center of photosynthesis mechanism. Divers studies have been carried out using cyanobacteria, green algae and high plants as materials. The research of pigment-protein complexes of red algae is far behind those of cyanobacteria, green algae and high plants. In red algae, studies are mainly in unicellular Porphyridium cruentum. Report on the structure and function of phycobilisomes of multicellular red algae is scarce. Research about phycocyanin (PC) and allophycocyanin (AP) is even less. Phycocyanin and allophycocyanin are the components of the phycobilisomes, and their content is low in phycobilisomes. Study of phycobilisome composition of red algae, is very meaningful to the deep understanding of the structure and function of phycobilisome and evolution of photosynthetic systems.This study focuses on the separation and purification method of phycobilisomes from P. urceolata and the properties and the characteristics of phycobiliprotein. A two step sucrose density ultracentrifugation method has been used to separate and purify the phycobilisomes of P. urceolata. This is an efficient method of P. urceolata phycobilisomes preparation. The phycobiliprotein, phycoeryanin (PE), PC and AP of phycobilisomes from P. urceolata were separated and purified by combanation of chromatographies and native polyacrylamide gel electrophores. Spectral properties, isoelectric point and strctural characteristics of phycobiliproteins were studied. The main contents and results of the study are as follows: 1. Separation and purification of phycobilisomes from P. urceolataThe use of ultrasonication, solubilization and two-step sucrose density gradient ultracentrifugation purified phycobilisomes. The results showed that solubilization with 2% NP-40 (final concentration, v/v) for 1.5 h and then a two-step ultra-centrifugation (138,000×g,3.5 h) with 1.0-2.0 mol/L sucrose density gradient first and 1.0 mol/L sucrose solution later can obtain phycobilisomes with highly pure (F675/F576=5.25) and complete. Diameter of the phycobilisomes is about 21 Onm.2. Purification of phycobiliprotein from phycobilisomesComparisonat different isolation and purification methods of R-PE found that Sephadex G-150 column chromatography in combination with ion exchange chromatography is effective for purifing phycoerythrin of phycobilisomes. A565/A280 of the purified R-PE is arrived at 4.95.R-PC was purified by native polyacrylamide gel electrophoresis with 7%(w/v) of separation gel (pH7.5) and 3%(w/v) of stack gel (pH5.5) after purified by the Sephadex G-150 column chromatography and ion exchange column chromatography. A618/A280 of the purified R-PC is arrived at 5.25.The elution conditions of ion exchange chromatography are that R-PE is eiuted by NaCl solution of 0-400 mmol/L linear gradient (25 mmol/L of PBS buffer preparation) with 500ml volumn and R-PC is eiuted by L NaCl solution of 50-400 mmol/L linear gradient (25mmol/L of PBS buffer preparation) with 500ml volumn. After R-PC was eluted from ion exchange column, AP can be isolated by native polyacrylamide gel electrophoresis with 5%(w/v) of separation gel (pH7.5) and 4%(w/v) of stack gel (pH5.5) from components which eluted by 400mmol/LNaCl andl.5mol/LNaCl from the ion exchange column respectively.3. Analysis of phycobiliprotein of phycobilisomesSpectral properties, isoelectric point, structure characteristic of purified R-PE, R-PC and AP have been researched. The results are as follows:The isoelectric point of R-PE is about pH4.7; molecular weight is 247kDa; R-PE contains four kinds of subunitsα,β,γ,γ'by SDS-PAGE with 13-21%(w/v) separating gel (pH9.0) and 4%(w/v) stacking gel(pH6.8). It has two different hexamer forms (α17.6β19.2)3γ29.5 (α17.6β19.2)3 and (α17.6β19.2)3γ31.0 (α17.6β19.2)3 and two hexamers have very close isoelectric points. The isoelectric points of R-PE subunits are pH5.0-5.8 and isoelectric points ofα/βsubunit are pH5.0 and pH5.8.The isoelectric point of R-PC is pH5.7. R-PC has three kinds of subunitsα,βandβ', has noγ-subunit and no colorless polypeptide by SDS-PAGE with 12%-21%(w/v) separating gel (pH8.8) and 4%(w/v) stacking gel (pH6.8). R-PC has two different hexamer forms (α17.5β21.3)6 and (α17.5β22.6)6. The isoelectric point ofα/βsubunit of R-PC is pH5.1 and pH5.2.The isoelectric point of AP is pH5.5. Results of SDS-PAGE with 13%(w/v) separating gel (pH 9.5),4%(w/v) stacking gel (pH6.8) show that AP has two kinds of chromospheres-carrying subunitsα,β. and quadratic AP1 (which dialyzed after second native-PAGE) and AP2 (which obtained by native-PAGE from fraction eluted by 400mmol/L NaCl) either have two colorless peptides. They all have two kinds of polymer form (α17β18.5)3L1, and (α17β18.5)3L2, AP3 (which obtained by native-PAGE from fraction eluted 1.5mol/L NaCl) has no colorless peptides. AP3 only has one type of polymer form(α17β18.5)3.Polymer form of phycobiliprotein of phycobilisomes was proposed in this study. It helps the understanding of the characteristic of phycobiliprotein and the structure of phycobilisomes of red algae and differences of phycobilisomes with cyanobacteria and unicellular algal. It can provide valuable research results for the evolution of algal chloroplasts, photosynthesis mechanism and micro-structure of photosystem.It also can provide theoretical basis for the application of marine red algae.