| Five kinds of purified phycocyanin PC-645(A645/A280>7)with different solubility in water are obtained by self-designed separation and purification process from laboratory cultured Chroomonas placoidea.And the difference of hydrophobicity and the relationship to subunit composition are quantitatively analyzed.The results show that there are probably two different forms of heterodimers in PC-645,namely,??1?1???2?1?and??1?1???2?2?.They may exist in different proportions and form various forms of aggregates with different solubility in aqueous solution,which will be precipitated under different ammonium sulphate saturation.The heterodimer containing?2 subunit unreported before shows higher hydrophobicity than the other one,and exists in 60%ammonium sulfate precipitation,which has the relatively weakest solubility in water?group 2?,in the highest percentage.While in 100%PC-645 with the strongest water solubility,only one heterodimer form of??1?1???2?1?appears?only component 1?.The water-soluble difference of PC-645 precipitates is probably due to the hydrophobicity difference of the heterodimers formed by different?subunits.Meanwhile,the method of sucrose density gradient centrifugation is explored and optimized,by which,two aggregation forms of PC-645 unreported previously are isolated,and are named as PC complex 1 and 2,respectively,after purification by ammonium sulphate precipitation and hydrophobic chromatography.The spectral characteristics,polypeptide composition of these two PC complexes as well as their particle morphological features under electron microscopy are measured and studied.The results show that the two purified PC complexes are free of fluorescent protein components except?and?subunits;PC complex 1 contain subunit?1,?1 and?2,?2,which is considered to be composed of two forms of heterodimers??1?1???2?2?and??1?1???2?1?;in PC complex 2,there's no?2 subunit is observed,and therefore probably be formed by only one kind of heterodimer??1?1???2?1?;The particle size of two complexes was about 20 nm under frozen electron microscopy and negative staining,indicating that phycocyanin of C.placoidea can form large molecular weight of aggregates on the basis of heterodimer,and the aggregate forms are not uniform.There are non-fluorescent protein bands in both high molecular weight area and 1018 kDa region in purified phycocyanin complex,whether it is a linker polypeptide remains to be further studied in the future.The exist states and formation conditions of macromolecular PC-645 complexes are investigated and compared to the corresponding conditions during ultra-high speed centrifugation,including centrifugal time,the gradients of sucrose,concentration of phosphate and pH.Accordingly,we conclude that the optimum conditions of phycocyanin complex separation from C.placoidea are:50 mmol/L phosphate buffer concentration,pH4.92 at algae broken stage;300 mmol/L phosphate buffer as sucrose density gradient solvent,pH 4.92,30000 RPM?BECKMAN Optima LXP preparative ultracentrifuge,SW41 Ti Rotor?,6 h during ultracentrifugation.On the basis of the experimental results described above,we suggest that the existential states of cryptophytic phycobiliproteins thylakoid lumen are not completely free and homogeneous,may exist not only in two or more kinds of herodimeric forms,but also further aggregate forms,which we call phycobiliprotein complex,on the basis of heterodimer.In the process of photosynthesis,cryptophytic phycobiliproteins can regulate the excitation energy transfer ratio from phycobiliproteins to the reaction centers embedded in the membrane and further regulate the photosynthesis process by the dynamic process of dissociation and aggregation.The theory provides a new idea and theoretical basis for understanding and studying the composition,characteristics and mechanism of photosynthetic systems in cryptophytic alga. |
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