Isolation，Purification And Sequence Difference Analysis Of PC645 Subunits From Chroomonas Placoidea

Four kinds of phycocyanin PC645 samples of Chroomonas placoidea were precipitated at 70%,80%,90%and 100%ammonium sulfate saturation respctiovely,and then purified to a ratio of A₆₄₅/A₂₈₀?7.Theαandβsubunits of PC645 were isolated and purified by SDS-PAGE;At the same time,the spectroscopic properties of these subunits were determined and analyzed.The characteristics and sequential differences of the twoβsubunits that are not clear at present are studied.The results showed that in addition toα1 andα2,PC645 contains two fluorescent bands with different molecular weights（17.8 kDa/15.9 kDa）and isoelectric points（pI 6.0/5.7）,which we named asβ1 andβ2 respectively.The content ofβ1 andβ2 showed a waxing and waning variation in the electrophoresis profiles as the increasing of ammonium sulfate saturation of different PC645 samples.This result indicates that the ratio of twoβsubunits in PC645 samples concern with its water solubility.αandβsubunits from purified 80%PC645 were isolated and purified by gel filtration chromatography,SDS-PAGE and IEF electrophoresis after urea denaturation.The spectral information,isoelectric point and pH tolerance of the subunits were also determined.The results indicated that the structure of PC645dimer is stable,PC645 with A₆₄₅=10 is completely denatured only after 48 hours of treatment by 8 mol/L of urea.The results of electrophoresis showed that theαandβsubunits after Sephacryl S-100 chromatography are completely purified,and PC645may contain two types ofβsubunits with different molecular weight and isoelectric point.Spectral analysis exhibited that the spectral activities are still maintained both in isolatedαandβsubunits.Theβsubunit was the terminal emission band,which produce the characteristic fluorescence of PC645 at 660 nm.Furthermore,the isolatedβsubunit maintained their spectral characteristics in PBS buffer during 45days of storage if preserved at pH 4.92.MBV chromophore inαsubunit displayed no fluorescence emission.And the isolatedαsubunit showed the best pH tolerance around pH 7.At the same time,the experimental process of large-scale separation of phycocyanin PC645 subunits by ion exchange chromatography was explored.PC645(A₆₄₅=10)treated with 8 mol/L urea for 48 h could not be combined with DEAE anion chromatography at pH 7;Under the condition of pH 8,the subunits could be combined with the chromatography agent,and three fractions with different electric charge amount could be eluted.However,the spectrogram showed that the protein tertiary structure of subuints separated under this condition were no longer complete,and the bilins could not transfer the absorbed energy orderely to the terminal bilin PCB（82）and produce the terminal emission.PC645(A₆₄₅=10)treated with 4 mol/L urea for 48 h could be successfully combined with DEAE anion chromatography agent at pH value of 7,and three fractions with different charge amount,which still maintain biological activity after resolution,could be eluted.The electrophoresis results showed that the experimental conditions of ion exchange chromatography performed a preferential separation of four PC645 subunits,which laid a foundation for large scale of pure subunit separation from PC645.The whole sequence of 177 amino acids including N-terminal of PC645β2subunit was obtained.There were 8 amino acid residues differences between our sequencing result and theβsubunit sequence that reported in 1990,and basically consistent with theβsubunit sequence（absent of 14 amino acid residues information in N-teminal in PC645）determined by crystal diffraction published by Stephen J.Harrop et al in 2014.Modeling method（SWISS-MODEL）and the ab initio prediction method（I-TASSER）were employed to predict the protein structure and function based on the complete amino acid sequence information ofβ1 andβ2subunits of PC645.The prediction results show that the spatial structure of the two subunits is different.The results above confirm that there are four different fluorescent subunits in PC645.Theβ2 subunit,which has not been reported previously,is different fromβsubunit generally recognized at present in terms of isoelectric point,molecular weight and amino acid sequence.Combined with the diversity characteristics of subunits and the difference of hydrophobicity of phycocyanin PC645,it is indicate that the existence of is not completely homogeneous in lumen.There may be two or more heterodimer forms ofα1α2β1β1,α1α2β1β2,α1α2β2β2.These conclude provide a theoretical basis on discussing the composition and properties of cryptophycobiliprotein subunits,and for the futher study of the photosynthetic system structure of cryptoalgae and the relationship with thier function of light harvesting.