【摘要】 以细胞色素C和溶菌酶为代表,研究了蛋白质在强亲和性金属螯合柱(IDA-Cu(Ⅱ))上的保留行为。考察了竞争剂、缓冲体系对蛋白质在IDA-Cu(Ⅱ)柱上保留值的影响。发现在PBS和NaAc-HAc缓冲体系用咪唑(Imid)和甘氨酸(Gly)作竞争剂,可使蛋白质得到较好分离;在Gly和Imid竞争体系,分别研究了pH值变化对蛋白质在IDA-Cu(Ⅱ)柱上保留值的影响。为使蛋白质在IDA-Cu(Ⅱ)柱上得到有效分离且减少固定金属Cu2+的流失,对Gly体系,最好采用竞争置换和降低pH值相结合的方式进行洗脱。对Imid体系,溶液pH值应控制在6.0为宜,低pH值下蛋白质则不宜被洗脱。结果表明,竞争剂浓度与蛋白质在IDA-Cu(Ⅱ)柱上的保留关系均符合计量置换模型。随着竞争剂浓度的增加,蛋白质的保留值减小。保留因子(k′)的对数与竞争剂浓度倒数(1/[D])的对数具有良好的线性关系,其线性相关系数分别在0.984和0.986以上。更多还原

【Abstract】 The retention behavior of proteins on strong affinity metal chelate column(IDA-Cu(Ⅱ) ) was studied using cytochrome-C(Cyt-C) and lysozyme(Lys) as typifiers.The effects of competitive agents and buffer systems on the retention of proteins on IDA-Cu(Ⅱ) column were investigated.In potassium dihydrogen phosphate(PB) and sodium acetate-acetic acid(NaAc-HAc) buffer systems,the better separation for proteins could be obtained by using imidazole(Imid) and glycine(Gly) as competitive agents.The effects of pH on the retention of proteins on IDA-Cu(Ⅱ) column were examined in Imid and Gly eluting systems,respectively.For effective isolation of proteins and less leakage of Cu 2+,the eluting method of competitive displace combined with decreasing pH was a better choice for Gly system.As for Imid,the pH value should be controlled at 6.0. Proteins could not be easily eluted at the low pH.The results indicate that the relationship between the concentration of competitive agent and the retention of proteins on IDA-Cu(Ⅱ) column conforms to the stoichio-metric displacement theory for retention(SDT-R) .The retention time decreased with increasing competitive agent concentration.The retention factor(k′) and the reciprocal of competitive agent concentration(1/D) were well logarithmic linear relationship.The linear correlation coefficients were above 0.984 and 0.986 respectively.更多还原