【作者】 肖庆敏；

【导师】 孙彦；

【作者基本信息】 天津大学 ， 生物化工， 2003， 硕士

【摘要】 本文研究了不同添加剂对促进变性牛碳酸脱水酶(CAB)的复性作用。考察了各种因素(添加剂浓度、pH值、温度和较低盐酸胍浓度下添加剂浓度)对复性收率的影响。并应用相关动力学模型,对酰胺化合物辅助CAB复性的动力学过程进行了深入分析,探讨了乙酰胺促进CAB复性的机理,试验了其它酰胺化合物对CAB复性的可能性。结果表明,乙酰胺、脲、硫脲、丙酮、β-CD、CTAB+β-CD 和Tween 40+β-CD均能有效促进变性CAB复性。β-CD、CTAB+β-CD 和Tween 40+β-CD随着浓度增大,促进作用增强;而乙酰胺、脲、硫脲均存在最佳浓度使变性CAB复性收率最大。乙酰胺、脲、硫脲、丙酮等添加剂不改变复性液的pH值,均可在较宽的pH范围(5.5~8.5)内促进CAB复性;添加剂的加入改变了CAB复性收率不随温度变化的规律,脲、乙酰胺在37℃、硫脲在45 ℃时复性收率最大。利用三级反应动力学方程拟和酰胺化合物辅助CAB复性的动力学过程。结果表明,模型计算值与实验数据能较好吻合。酰胺化合物与盐酸胍类似,复性速率常数 KN值大于没有添加剂时的自发复性,但随添加剂浓度增加而降低,聚集体生成速率常数KA值则在最佳添加剂浓度下出现最小值,从而使KN/KA值最大,复性收率r值最大。降低复性液中盐酸胍浓度,适当提高添加剂浓度,乙酰胺和脲仍可使变性CAB得到同样的复性效果;而硫脲必须在一定浓度的盐酸胍存在下,才能有效促进CAB复性。由于高浓度乙酰胺会引起蛋白质的不可逆变性,因此乙酰胺虽可用做辅助蛋白质复性的有效添加剂,但不能作为复性蛋白质的变性剂使用。为了进一步探明乙酰胺辅助变性CAB复性的作用机理,根据上述结果进一步对类似化合物如含有酰胺基团的甲酰胺、丙酰胺、N,N-二甲基甲酰胺、N,N-二甲基乙酰胺促进变性CAB的复性作用进行了研究,发现它们均能有效地促进CAB复性,无论从其辅助CAB复性的动力学特性,还是随pH和温度的变化规律都与乙酰胺相似,因此认为酰胺基团对蛋白质复性的促进作用具有较大的贡献。更多还原

【Abstract】 The role of various additives on the refolding of denatured bovine carbonic anhydrase Ⅱ(CAB) was investigated. The effects of additive concentration in two different GdmCl concentrations、pH of the solution and temperature were studied. The kinetics of CAB refolding assisted by amides were analyzed, the principles of CAB refolding assisted by acetamide discussed and the possibility of other amides as additives was discussed and experimented. It was indicated that the denatured CAB could be properly refolded in the presence of acetamide, urea, thiourea, acetone, β-CD, CTAB+β-CD and Tween 40+β-CD. The refolding yield of CAB was improved as the concentration of β-CD, CTAB+β-CD and Tween 40+β-CD increased, while there was an optimal additive concentration for CAB reach its highest yield when acetamide, urea, thiourea were used. The yield can be improved at a wide pH range when acetamide, urea, thiourea and acetone existed. The optimal temperature were 37, 37 and 45 ℃for aetamide, urea, thiourea respectively. The kinetic model was in good agreement with the experimental results. The mechanism of CAB refolding assisted by amides were analogous to that of GdmCl. The kN, bigger than that of control, decreases with increasing of additive concentration, while there existed an optimal additive concentration minimizing the aggregating rate constant(kA) and maximum kN /kAwhich leads to the maximum recovery of CAB. When lowering the concentration of GdmCl to 0.0625 mol·L-1, the refolding yield could be the same as that in higher GdmCl concentration (0.125 mol·L-1) if the acetamide or urea concentration in the refolding buffer were a little increased. However, thiourea should always be used in high GdmCl concentration due to the unsufficient depressing effect of thiourea on the CAB intermolecular interactions.The possibility of acetamide as a denaturant was tested, it was found that acetamide in high concentration lead to the unconvertible denaturation of CAB although it was a good additive.CAB renatuaration could also be assisted by other amides, such as formamide, propamide, etc, with which the change of CAB refolding yield with time, pH of the buffer and temperature were similar to that of acetamide.更多还原