Isolation And Purification Of Phycoerythrin From Corallina Officinalis And Its Characteristic

Large numbers of alga were collected from littoral of Fujian province. By the absorption spectra scan (400~700nm) of crude extraction, we found three kinds of red alga that only contain R-phycoerythrin from the collected alga. After compared the other characters of three kinds of red alga, we choose Corallina officinalis as our sample, which is abundant and can grow in any season.By a method using fraction precipitation of ammonium sulphate, ion exchange, modified hydrorylapatite and gel filtration chromatography, we separated and purified two kinds of phycoerythrin(R-PE and r-PE) from Corallina officinalis . The purity standard(ratio of ODs65/OD2go) of R-PE reaches to 11, which is the highest purity comparing with the reported purity of phycoerythrin. The results of physical and chemic characteristic show that the molecular weight of R-PE is 194kD, which is differ from the other reported R-phycoerythrin's (240kD) . The molecular weight of subunits were determined . The molecular weight of a and P subunits are alike for about 20kD. The molecular weight of y subunit is about 31 kD.The subunit composition of R-PE in Corallina officinalis is presumably (a p ) 4 y , which is differ from (a P ) 6 y that is reported by other researcher. Moreover, we must to do more other work to prove if it contains the fourth subunit. Comparing the physical and chemic characteristic between R-PE and r-PE, we found that Y is absent in r-PE. As a result, their maximal absorption wavelength in visible absorption spectra is discrepant to lOnm. Their fluorescence spectra in excitation and emission wavelength and emission intensity is differ from each other. By comparatively discussing, we conjectured the possible function of r-PE in structure of phycobilisome. Based on the method of BCA, we menstruate the molar extinction coefficient of R-PE. The molar extinction coefficient in the wavelength of 565nm and 280nm is 4.75X K^mor'cm"1 and 4.07 X104morIcm"1 respectively. We prepared the antiserum of R-PE and determined its antiserum dilution. At the same time, we evaluate the specialization of this antiserum by Western-blot.Cloning and sequencing of the genes coding a and 3 subunit of phycoerythrin of a red alga-Corallina officinalis are reported firstly(GenBankaccession number: AF510986). The sequence contain 1,140 nucleotide, including 10 nucleotide that locate in the upstream of the initiation codon ofP subunit, 534 coding for P subunit(coding 177 amino acid), 101 nucleotide for gene spacer and 495 nucleotide that draw near the 5' terminal of a subunit(coding 164 amino acid). We comparatively analyze sequence characteristic and similarities in the nucleotide and in amino acid level with nine known red alga PE genes. Based on the analytic result, we consider that a and P subunit can be regard as one of taxonomic standard in molecule biology level. In addition, we determined the N-terminal amino acid sequence of Y subunit and can't find the similar sequence in known protein.Using SPDP as a heterobifunctional crosslinker, R-PE isolated from Corallina officinalis and sheep anti-rabbit IgG purified by ion exchange chromatography as conjugated reagent, we prepared the phycoerythrin fluorprobes by two-step binding protocol. We cited nylon membranes as our solid-phase medium, which is often used as solid-phase medium in dot immuno-enzyme filtration assay. At the same time, we also cited reagent and protocol that is used in dot-ELISA. At last, we used this fluorprobes to detect Tobacco mosaic virus by two fluorescence immunoassay. Furthermore, we compared the sensitivity of four different. At the end of this article, we deeply discussed how to improve the sensitivity of immunoassay that used the phycoerythrin fluor as probes.