Studies On Purification, Identification And Protein Characteristics Of Ginkgo Vegetative Storage Proteins

Ginkgo, as special plant resources in China, has high development potential. Ginkgo vegetative storage proteins (VSPs) was the main form of storage nitrogen during overwinter period. VSPs played an important roles in reducing the loss of the nutrients in ginkgo tree during the winter, biomass accumulation and the ability of resisting adverse environment. There were studies on cytology observation and biochemical properties of Ginkgo VSPs by some scholars on, and the molecμlar weight of the protein as 32KD and 36KD have been confirmed as the main VSPs of ginkgo.But the purification and structure-activity of ginkgo VSPs have been rarely studied. This research based on the previous research, studyed on the extraction and purification technology of the 32KD and 36KD Ginkgo VSPs; and then, biloba mass spectrometry and characteristics of Ginkgo VSPs were studied. The major elements were as follows:Tris-HCl was confirmed as the best extract solution.And the resμlt of single factor experiment and Uniform Design shows that the optimum extraction conditions:extraction time as 10h, extraction pH 9.0, extract concentration of 0.25 mol/L, liquid-solid ratio as 10:l;and the protein extraction rate reach 70.22% under these conditions.The result of SDS-PAGE showed,the proteins extracted from the branches were VSPs.DEAE-Sepharose FF was used for crude protein preliminary separation The influence of sample concentration, sample volume,velocity and ion gradient have considered to separate.And the optimized chromatographic parameters have been confirmed as:sample concentration: 10mg/ml, sample volume:5ml, sample flow rate: 1ml/min, ion gradient: 0-0.3mol/LNaCl.Ginkgo VSPs were separated into 4 components:Ⅱ,Ⅱ,Ⅲ,Ⅳ..The result of SDS-PAGE showed 32KD and 36KD ginkgo VSPs were contained in component IV. ComponentⅣwas collected to for Sephadex G-75 gel separation. The influences of sample concentration and velocity have considered to separate. The optimized chromatographic parameters were:sample concentration: 15mg/ml, sample volume:5ml, sample flow rate:0.3 ml/min.ComponentⅣwas separated into 3 components. The result of SDS-PAGE showed that the target protein 36KD ginkgo VSPs which was contained in componentⅡhas reached electrophoresis pure.Biochemistry and characteristics of the 36KD ginkgo VSPs were studied.The protein was a acidic protein which isoelectric point was about 4.and the protein solubility will increase when the solution was alkaline.But the color of protein will deepen because of maillard reaction when the pH greater than 9.0. The study on the influence of NaCl to protein showed that, the protein solubility will decrease when the NaCl concentration higher than 4%.By DSC,we found the denaturation temperature of 36KD ginkgo VSPs was 87.7℃which need the power of 205.6J/g.LC-MS resμ1t showed that 36KD ginkgo VSPs a new type of VSPs.By PMF, peptide RMNTGYGARTPEVKC from 36KD ginkgo VSPs was found that it was had similar structure domain to tuber storage protein of Dioscorea polystachya. According to this, we can conjecture that 36KD ginkgo VSPs had the same activity to acid phosphatase which was played an important role in Organophosphorus enrichment;the other peptide KGNAGSLSINRVAYQLKRI was found that it was had similar structure domain toalpha_CA_prokaryotic_like which was played an important role in Photosynthesis.The result of FT-IR showed:36KD ginkgo VSPs has Special functional groups such as -NO2,-Br;andβ-folding was the main formal on the Secondary structure of the 36KD ginkgo VSPs.