聚丙烯酸分离纯化苦瓜种仁碱性蛋白的方法及影响因素

The Method and Influencing Factors of Alkalescency Protein Purification from Bitter Melon (Momordica charantia) Seeds by Polyacrylicacid Precipitation

摘要:

以苦瓜籽为材料，研究了聚丙烯酸分离纯化苦瓜种仁碱性蛋白的方法及影响因素。等电点沉淀试验表明，柠檬酸、盐酸分别调节苦瓜种仁粗提液pH至6.0、4.0时，各有14.62%和32.49%的苦瓜种仁蛋白被沉淀。醋酸的等电点沉淀作用呈现阶段性特点，pH 6.0和4.0时分别有26.17%和38.72%的苦瓜种仁蛋白被沉淀。醋酸、盐酸和柠檬酸处理的1mL苦瓜种仁粗提液（pH 4.0），1%PAA选择性沉淀碱性蛋白（等电点pI为8.65～9.30）的最佳用量分别为100μL、120μL和100μL。醋酸调节苦瓜种仁粗提液pH分别至5.0、4.0和3.0，等电点沉淀后的上清液用PAA沉淀碱性蛋白，当PAA（1%）用量为160μL/mL提取液时，pH5.0和3.0样液分别有33.77%和43.56%蛋白质被沉淀；当PAA用量为120μL/mL提取液时，pH 4.0样液中30.83%蛋白质被沉淀。PAA-蛋白质复合物溶解于碱性溶液（pH＞9.0），当溶液NaCl浓度为3.0%时，溶液蛋白质浓度最高。PAA选择性沉淀的苦瓜种仁碱性蛋白经Sephadex G-75柱层析分离，分别在175min和300min出现主峰Ⅰ和Ⅱ。SDS-PAGE和IEF分析表明主峰Ⅰ的分子量约为30kD，pI值约为9.5，主峰Ⅱ的分子量约为10kD，pI值约为9.3。

Abstract:

In this study, polyacrylicacid precipitation alkalescence protein from Momordica charantia L. seeds was studied, and the effect of conditions on experiment was also evaluated. Isoelectric precipitation is achieved by adjusting the pH of a protein solution and is based on that a protein's solubility is at minimum at its pI. The sample was titrated to pH 6.0 with citric acid, and 14.62% proteins were precipitated. With hydrochloric acid to pH 4.0, 32.49% proteins were precipitated. With the acetic acid to pH 6.0 and pH 4.0, 26.17% and 38.72% proteins were precipitated, respectively. In the 1 mL Bitter melon seeds extraction(pH 4.0) adjusted by acetic acid, hydrochloric acid and citric acid, the optimum dosage of PAA（1%）precipiting alkalescency protein (pI 8.65～9.30）was 100μL, 120μL and 100μL respectively. The respective extraction (1mL) 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 （1%） 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 （1%） was 120μL/mL, the protein decreased in the supernatant was 30.83% at pH 4.0. PAA-Protein complex could redissolve in alkaline conditions (pH＞9.0) and the protein most easilly redissolved when the NaCl was 3.0%. The bitter melon seeds extraction after PAA purification flowed throμgh the Sephadex G-75 columns. The peaks Ⅰand Ⅱ were obtained after 175min and 300min, respectively. SDS-PAGE and IEF analysis showed that the molecule weight from peaks Ⅰwas 30kD with pI 9.5, peaks Ⅱ10kD with pI 9.3.