| Phycocyanins(PC645)with different solubility were isolated and purified from a cryptophytic alga Chroomonas placoidea T13 cultured in lab.The relationship of hydrophobicity difference and subunit composition were quantitatively analyzed.The properties,composition as well as the microstructure of aggregated form of PC645obtained by using our established method were investigated.Phycocyanins were fractionated precipitated under 70%,80%,90%and 100%ammonium sulfate from C.placoidea,and were purified by two steps of G-100 gel filtration chromatography.The pure PC645 samples(A645/A280?7)with similar molecular weight of heterodimers were obtained.SDS-PAGE analysis showed that different ratios of?2subunit(15.9 k Da),which has not been reported previously,was observed in purified PC645 in addition to?(8-10 k Da)and?1(17.8 k Da)subunits.The?2subunit,whose content diminishing accompanied by the increasing of solubility of PC645 samples,was more enriched in 70%and 80%PC645 that therefore may exist in(?1?2?1?2)form of heterodimers,or(?1?2?1?1)mixed with(?1?2?1?2)and(?1?2?2?2);However,in 90%and 100%PC645 with stronger solubility in aqueous solution,?2subunit was minimized or almost absent,suggesting that these PC645 exist mainly in the form of(?1?2?1?1).This result indicated that the varieties of solubility or hydrophobicity of PC645 samples are due to the content deference of?2subunit.The results of two dimensional electrophoresis showed that the 100%PC645created three bands of p I=8.9,p I=6.7 and p I=5.9 during denatured IEF.70%PC645had another fourth band of p I=5.7,accompanied with a series of blue bands around p I4?6.3.SDS-PAGE confirmed that the two green bands of p I=8.9 and p I=6.7 were rich in?1and?2,respectively;the blue bands of p I=5.9 and p I=5.7 were rich in?1and?2.The results indicated that the two?subunits differed not only in molecular weights,but also in isoelectric points.Different proportions of?1and?2in 70%PC645 caused the appearance of blue bands at p I 4?6.3;While,the absent of?2subunit was corresponding to the disappearance of blue IEF bands except the?1subunit in 100%PC645.The sucrose density gradient centrifugation method for separation of PC645complexes was established and primarily optimized.PC complexes showed similar absorption and fluorescent features with PC645 heterodimers.They were primarily consisted of two kinds of?subunits and?1subunit,as well as traces of?2subunit,which allowed us to suppose that they were aggregates of(?1?2?1?1)heterodimers predominantly.In addition,two non-fluorescent protein bands around 13 k Da and 60k Da were also found.Further investigations are need for identifying whether or not linker peptides they are.The obtained PC complex migrated at the similar position as that phycoerythrin hexamers(240 k Da)did during sucrose density gradient centrifugation.The negative staining and electron cryo-microscopy measurement show that the PC645 heterodimers were irregular pellets less than 10 nm in size;however,the PC complexes were homogenous,geometrical particles with an average size of 20 nm.For the first time we observed that the shape of PC complexes was hollow ring.The results above indicated that cryptophytic phycobiliproteins were able to form aggregates with higher molecular weight on the basis of heterodimers.The average molecular weight of PC complexes was about 240 k Da,which were consisted of 4 or 5 PC645 heterodimers.The heterogeneity of two?subunits in PC645 and the existence of aggregated phycocyanin complex have broken the previous understanding of the existence state of cryptophytic phycobiliproteins,and provide a new idea and theoretical basis for further discussing the existence states of cryptophytic phycobiliproteins,as well as the structure and mechanism of photosynthetic systems. |
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