Polyelectrolyte As Vehicles For Isolation And Purification Of Alkalescence Protein And The Conditions Optimization Of Alkalescence Peptides SDS-PAGE

Both isoelectric and polyelectrolyte precipitation have been shown to be effective and surprisingly selective in separating proteins from the extracts of various hosts. Because it is a simple unit operation, relatively inexpensive and straightforward to be scaled-up, precipitation is often used in the early stages of protein purification processes for protein recovery and purification. In this study, Polyacrylic Acid (PAA) was used to isolate and purify alkalescence protein from Momordica charantia Zseeds and alkalescence peptides from bee venom.Besides, the effect of acetone on fat and analysis of alkalescence peptides by SDS-PAGE were also evaluated in this paper.Acetone and Momordica charantia L. seeds (bitter melon seeds) or bee venom were homogenated, filtrated and washed (with acetone). The acetone powder was obtained by evaporation. Compared to water extraction, protein by acetone extraction was much water-soluble with little fat, which contributed to recovery and purification of protein.By the isoelectric precipitation, the neutral and acidic protein from Momordica charantia L. seeds was removed at the pH 7.0~3.0. In the upper solution, the kinds and content of the alkalescence protein with pI above 8.65 was the same to the extraction. The alkalescence protein with pI 8.65 decreased with pH falling. The isoelectric precipitation was related to acid medium. When the Momordica charantia L. seeds extraction was adjusted by the citric acid, the isoelectric precipitation was obvious during the range of pH7.0～6.0, and 14.62% protein precipitated at pH6.0. When the extraction was adjusted by the hydrochloric acid, the isoelectric precipitation was obvious during the range of pH7.0～4.0, and 32.49% protein precipitated at pH4.0. When the extraction was adjusted by the acetic acid, the isoelectric precipitation was obvious during the range of pH7.0～6.0 and pH5.0～4.0, and 26.17% and 38.72% protein precipitated at pH6.0 and pH4.0, respectively.Precipitation of PAA with alkalescence protein from Momordica charantia L. seeds was influenced by acid medium, PAA concentration and pH. In the solution (1 mL) adjusted by acetic acid, hydrochloric acid and citric acid, PAA (1%) was added at 100μl,120μl and 100μl. SDS-PAGE showed that PAA precipitation protein in extraction adjusted by acetic acid was more efficient than hydrochloric acid and citric acid. The extraction was titrated to pH 5.0. pH 4.0, and pH 3.0 by acetic acid. After isoelectric precipitation, the PAA precipitation protein was performed. When concentration of PAA was 160μl/mL, the protein decreased in the supernatant was 33.77% at pH 5.0 and 43.56% at pH 3.0. When concentration of PAA was 120μl/mL, the protein decreased in the supernatant was 30.83% at pH 4.0. PAA -Protein complex counld redissolve in alkaline conditions(pH>9.0)and the solubility was related to NaCl concentration. When the NaCl was 3.0%, the protein most.easilly redissolved.The bitter melon seeds extraction after PAA purification flowed through the Sephadex G-75 columns. The peaks I and II were obtained after 175min and 300min, respectively. SDS-PAGE and IEF analysis showed that the molecule weight from peaks I was 30kDa with pI 9.3, peaks I 10kDa with pI 9.5.The bee venom extraction after PAA purification flowed through the Sephadex G-25 columns. Two peaks were obtained. Their molecule weights were about 2800Da and 1000Da. IEF showed that peak I included the protein with p/9.0.SDS-PAGE with urea and high cross lingking (C, C≥6.0%) has been used to separate and examine the alkalescence peptides. Because of its characteristics of molecule weight and charge, the polyacrylamid electrophoresis in acid environment is the method accustomed. This study evaluated the effect of SDS-PAGE with the low C separating alkalescence peptides under the different conditions.SDS-PAGE showed that, the low molecule weight peptides in samle and marker accumulated in the front of the gel with the C 0.67%. When the value of C was 1.20%, though the low molecule weight peptides in samle were not still separated, the peptides (Mr≤16950Da) in marker appeared clear. When the C was 1.55%, the peptides in both were separated efficiently. When the C was up to 2.00%,the bands were little visually. When the C was above 3.00%, the peptides couldn't be separated.