Structural And Functional Investigation Of Phycocyanin From Chroomonas Placoidea

In this thesis，we discussed the isolation and purification, assembly of structure,active conformation, energy transfer and antioxidant activity of phycocyanin645(PC-645) from Chroomonas placodiea T13. The main results are as follows:Combined with two-dimensional electrophoresis, protein blotting, N-terminalsequencing and mass spectrometry analysis，the existence of a new fluoresecentsubunit—β2from PC-645was confirmed. β2subunit has a smaller molecular weight(18.5kDa) and lower PI (pH5.7) than that of reported β1subunit. The results of massspectrometry showed that the difference between β2and β1may exist in the sequenceof β16~30and β79~84; SDS-PAGE analysis showed that the PC-645precipitated bydifferent concentration of ammonium sulfate contained different proportions of α andβ subunits. Hydrophobic fractions contained more higher proportions of β2subunit; alarger agreggeted PC-645complex was isolated by sucrose density gradientcentrifugation, which showed a different peptide composition compared withdissociated PC-645.Secondary structure analysis demonstrated that there were about28%α-helicesand15%β-sheets in natural PC-645. PC-645showed a high structural flexibility andexihibited both structural and functional stability in wide range of pH and certainconcentration of urea monitored by synchronous dynamic fluorescence, absorbanceand UV-CD spectra during denaturation and renatuaration; We conclude thatpH-induced conformational and functional dynamics of PC-645could be divided intothree regions: stable region (pH3.5~7), metastable region (pH7~10) and unstableregion (pH<3or pH>10). PC-645was more stable in acid condition than in alkalicondition. This result may be in compliance with its distinct environment andphysiological function.Whole-band of CD spectrum of PC-645in190nm~750nm was obtained andresolved for the first time. The four absorption peaks of PC-645:540nm (shoulder peak),582nm,625nm (shoulder peak) and645nm, were considered to correlated toDBV、MBV、PCB (β158）and PCB (β82）, respectively. DBV/MBV and PCB (β158)/PCB (β82) consisted as two exciton-coupled bilin pairs. Energy transfer withinexciton-coupled pairs was by exciton splitting, while between exciton-coupled pairsby F rster resonance; Asp played an important role in the electron transfer betweenthe exciton-coupled pairs of bilins; Tyr might be grouped with PCB (β82) in aconservative hydrophobic microenvironment, and made the absorption peak of PCB(β82) red shifted from610nm to645nm because of the conjugation of hydrogen bond.PCB (β82) was most likely the final receptor of energy transfer inside PC-645.Compared with denatured PC-645, natural PC-645dimers exihibitedanti-oxidation activity, which colud scavenge the free radical of oxygen (O2－), andshowed different inhibition activity when acted with different kinds of free oxygenradical.