变性核糖核酸酶在疏水表面置换吸附焓的微量热研究
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摘要
本文利用高精密度的MicroDSC-Ⅲ生物微热量计测定25℃不同变性剂浓度变性核糖核酸酶A(RNase A)在疏水表面的置换吸附焓变(△H),按液/固吸附计量置换理论(SDT-A)对置换吸附焓变进行细分,同时结合摇床法测得的相应条件下吸附等温线数据,探讨各部分在吸附过程中的热力学贡献。同时配合傅立叶变换红外光谱(FTIR)、和Sapphire差示扫描量热(DSC)方法对相应条件下在疏水表面上吸附态RNase A进行的热扫描结果,综合分析RNase A在PEG-600表面疏水吸附折叠规律。
同溶液中蛋白相比,吸附到PEG-600疏水表面后的吸附态RNase A稳定性降低。同一变性条件下的吸附态RNase A,较高表面覆盖度时的稳定性较高。表面覆盖度相同时,变性剂浓度越大,其吸附态的热稳定性越好。在天然和低变性剂浓度下,蛋白可能发生二级结构的丢失。
随着盐酸胍浓度的增加,△H值先增大后减小。我们认为天然RNase A的放热效应是由于构象丢失过程伴随的水合作用所致,也就是ΔΗmh放热效应在总焓变ΔΗ中起主要作用;然而,RNase A在0.3 mol·L~(-1) GuHCl时表现为较大的吸热效应则是由构象丢失的诱导所致,也就是ΔΗml吸热效应在总焓变?Η中起主要作用;同一GuHCl浓度下,不同初始蛋白浓度时△H值的测定表明,天然RNase A的吸附是一个放热过程,这是因为天然吸附态蛋白发生了一些有序结构丢失,是构象丢失过程中伴随的水合作用ΔΗ_(mh)对吸附热效应起主导作用,同时天然蛋白吸附量都为负值,说明此条件下水合作用明显,溶液中的水分子以水合的形式结合到蛋白分子和填料的表面,这也与△H〈0相吻合;经0.3 mol·L~(-1) GuHCl变性的RNase A的置换吸附热效应△H在实验条件下均为正值,并随蛋白浓度的增加而降低,表明构象丢失和去水合作用(均为吸热)强于吸附亲和作用和构象丢失过程所伴随的水合作用(均为放热)。
The MicroDSC-Ⅲcalorimeter was used to measure the displacement adsorption enthalpy of Ribonuclease A (RNase A) which originally had been denatured by various concentrations of guanidine hydrochloride (GuHCl) mixing with moderately hydrophobic PEG packings in 1.5 mol.L-1 ammonium sulfate ((NH4)2SO4) salt solutions at 25℃. According to the△H which was divided into four enthalpy fractions by stoichiometric displacement theory (SDT) and adsorption isotherm data, the thermodynamic contribution of enthalpy fractions was discussed. The adsorption folding law also was analyzed by CD、FTIR and DSC.
The thermal stability of adsorbed RNase A was decreased comparing with in the solution and adsorbed RNase A was more stable in higher surface coverage or higher GuHCl concentrations when the other factor is fixed. Conformational loss may occurred on adsorbed RNase A at zero and lower concentrations of GuHCl.
The△H values increase first and then decrease with the increment of GuHCl concentration whether the concentrations of adsorbed RNase A was 0.4、0.7 or 1.0 mg.ml~(-1). An exothermic effect for native RNase A is mainly attributed mainly to hydration associated with conformational loss, based on the thermal analysis of enthalpy fractions of protein adsorption, it was expressed asΔΗmh in the enthalpy fractions, while a great endothermic effect for 0.3 mol/L GuHCl is induced by conformational structure loss which was denotedΔΗml in the enthalpy fractions.
The measured△H values of RNase A in the same GuHCl concentrations and various RNase A concentrations showed that the adsorption process of native RNase A is exothermic. That’s because conformational loss occurred on adsorbed native protein and hydration associated with conformational loss (ΔΗ_(mh)) were predominant for the endothermic effect. The adsorbed amount is negative under this condition, so the water molecules were obviously combined with the protein molecules and packings on the surface, which was identical with the negative△H. The△H values of denatured RNase A by 0.3 mol/L GuHCl are all positive and decrease with increase of surface coverage, indicating that conformational loss and dehydration may be prior to adsorption affinity and hydration associated with conformational loss.
同溶液中蛋白相比,吸附到PEG-600疏水表面后的吸附态RNase A稳定性降低。同一变性条件下的吸附态RNase A,较高表面覆盖度时的稳定性较高。表面覆盖度相同时,变性剂浓度越大,其吸附态的热稳定性越好。在天然和低变性剂浓度下,蛋白可能发生二级结构的丢失。
随着盐酸胍浓度的增加,△H值先增大后减小。我们认为天然RNase A的放热效应是由于构象丢失过程伴随的水合作用所致,也就是ΔΗmh放热效应在总焓变ΔΗ中起主要作用;然而,RNase A在0.3 mol·L~(-1) GuHCl时表现为较大的吸热效应则是由构象丢失的诱导所致,也就是ΔΗml吸热效应在总焓变?Η中起主要作用;同一GuHCl浓度下,不同初始蛋白浓度时△H值的测定表明,天然RNase A的吸附是一个放热过程,这是因为天然吸附态蛋白发生了一些有序结构丢失,是构象丢失过程中伴随的水合作用ΔΗ_(mh)对吸附热效应起主导作用,同时天然蛋白吸附量都为负值,说明此条件下水合作用明显,溶液中的水分子以水合的形式结合到蛋白分子和填料的表面,这也与△H〈0相吻合;经0.3 mol·L~(-1) GuHCl变性的RNase A的置换吸附热效应△H在实验条件下均为正值,并随蛋白浓度的增加而降低,表明构象丢失和去水合作用(均为吸热)强于吸附亲和作用和构象丢失过程所伴随的水合作用(均为放热)。
The MicroDSC-Ⅲcalorimeter was used to measure the displacement adsorption enthalpy of Ribonuclease A (RNase A) which originally had been denatured by various concentrations of guanidine hydrochloride (GuHCl) mixing with moderately hydrophobic PEG packings in 1.5 mol.L-1 ammonium sulfate ((NH4)2SO4) salt solutions at 25℃. According to the△H which was divided into four enthalpy fractions by stoichiometric displacement theory (SDT) and adsorption isotherm data, the thermodynamic contribution of enthalpy fractions was discussed. The adsorption folding law also was analyzed by CD、FTIR and DSC.
The thermal stability of adsorbed RNase A was decreased comparing with in the solution and adsorbed RNase A was more stable in higher surface coverage or higher GuHCl concentrations when the other factor is fixed. Conformational loss may occurred on adsorbed RNase A at zero and lower concentrations of GuHCl.
The△H values increase first and then decrease with the increment of GuHCl concentration whether the concentrations of adsorbed RNase A was 0.4、0.7 or 1.0 mg.ml~(-1). An exothermic effect for native RNase A is mainly attributed mainly to hydration associated with conformational loss, based on the thermal analysis of enthalpy fractions of protein adsorption, it was expressed asΔΗmh in the enthalpy fractions, while a great endothermic effect for 0.3 mol/L GuHCl is induced by conformational structure loss which was denotedΔΗml in the enthalpy fractions.
The measured△H values of RNase A in the same GuHCl concentrations and various RNase A concentrations showed that the adsorption process of native RNase A is exothermic. That’s because conformational loss occurred on adsorbed native protein and hydration associated with conformational loss (ΔΗ_(mh)) were predominant for the endothermic effect. The adsorbed amount is negative under this condition, so the water molecules were obviously combined with the protein molecules and packings on the surface, which was identical with the negative△H. The△H values of denatured RNase A by 0.3 mol/L GuHCl are all positive and decrease with increase of surface coverage, indicating that conformational loss and dehydration may be prior to adsorption affinity and hydration associated with conformational loss.
引文
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