The Isolation,Purification And Characteristic Analysis Of R-phycocyanin From Polysiphonia Urceolata Grev

Phycobiliprotein was extracted from the fresh Polysiphonia urceolata Grev in 50 mmol·L-1 phosphate buffer,pH 7.0.After the phycobiliproteins were ultrafiltrated with 500 kDa and 50 kDa membrane capsules,respectively.The concentrated phycobiliprotein concentrate was isolated by gel filtration using Sephacryl S-300 and Sephacryl S-200 to give R-PC/AP fractions with characteristic absorption peaks at 618 nm and 650 nm.R-PC/AP fractions were isolated by Q Sepharose Fast Flow ion exchange chromatography with NaCl salt concentration gradient elution.The pH gradient of 7.5～6.5 is added to the elution process.The R-PC and AP fractions were collected respectively.After that,the R-PC and AP fractions obtained by ion exchange chromatography were purified by Phenyl Sepharose 6 Fast Flow hydrophobic chromatography.The column was eluted with the concentration gradient of PBS salt concentration and the R-PC,R-PC2 and AP fractions were collected.The R-PC fraction obtained by the chromatography display single band in native polyacrylamide gel electrophoresis.The results of native polyacrylamide gel electrophoresis showed that the R-PC2 fraction contained a small amount of R-PE.The isoelectric points of the R-PC and R-PC2 fractions were measured at 5.63 and 5.62 by native isoelectric focusing electrophoresis.According to the results of denatured polyacrylamide gel electrophoresis,both R-PC and R-PC2 fractions contain a a subunit and two β subunits.The relative molecular mass of the a subunit is 18.3 kDa,and the relative molecular mass of two(3 subunits are 20.5 kDa（β20.5）and 21.4 kDa（β21.4）.But the relative content of two kinds of β subunits is different.The isoelectric points of two βsubunits of the R-PC fraction are 5.8（p5 8）and 5.74（β5.74）from denaturation isoelectric focusing electrophoresis.The isoelectric point of two p subunits of R-PC2 fractions are 5.8（β5.8）and 5.75（β5.75）.Two dimensional electrophoresis showed that the four β subunits of phycocyanin were β_21.4^5.8、β_20.5^5.8、β_21.4^5.74 β_20.5^5.74.