荧光光谱法研究氨基改性介孔泡沫对DhaA的稳定化机理
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摘要
采用F-4600荧光光谱仪,对尿素和二甲基亚砜两种变性剂中烷基卤脱卤酶DhaA在氨基改性介孔泡沫固定化前后的荧光光谱特征进行测定。运用荧光相图分析DhaA在两种变性剂中的去折叠过程,并结合活性残留率进行了变性过程热力学参数计算,比较固定化前后DhaA去折叠过程和热力学参数的区别。实验结果表明, DhaA催化活性随变性剂浓度增加而降低。相同变性剂浓度下,固定化DhaA能够比游离态DhaA保持更高的催化活性,在变性剂到达临界浓度之前(尿素浓度5.5 mol·L~(-1), DMSO浓度7 mol·L~(-1)),氨基改性介孔泡沫的稳定化作用显著。DhaA在尿素诱导下的变性过程符合"二态模型",而在DMSO诱导下符合"三态模型", DhaA中间态出现在浓度为5.6 mol·L~(-1)。氨基改性介孔泡沫固定化不改变DhaA变性过程,但能够提高DhaA的去折叠热力学参数。在尿素诱导下,计算得到的DhaA初始吉布斯自由能变ΔG(H_2O)为8.51 kcal·mol~(-1),固定化后ΔG(H_2O)提高为9.55 kcal·mol~(-1);但由于尿素分子容易通过静电作用进入氨基介孔泡沫孔道,固定化后DhaA的溶液可及面积m由3.69 kcal·(mol·mol·L~(-1))~(-1)增大到4.00 kcal·(mol·mol·L~(-1))~(-1),孔道内的氨基、羟基能够通过氢键作用增强DhaA的刚性,从而有效的降低了尿素可及面积增加带来的影响,提高了DhaA的尿素耐受性。在DMSO诱导下,计算发现游离态与固定化DhaA在折叠态向中间态转变过程中的ΔG(H_2O)均为12.12 kcal·mol~(-1),由于孔道内的氨基、羟基能够有效阻碍非极性DMSO分子的进入,造成m从3.39 kcal·(mol·mol·L~(-1))~(-1)降低为2.30 kcal·(mol·mol·L~(-1))~(-1);当DhaA从中间态向去折叠态转化时, DhaA内部疏水基团暴露导致m增加,由于孔道内极性微环境作用,固定化DhaA的m值(4.40 kcal·(mol·mol·L~(-1))~(-1))仍然低于游离态DhaA(4.94 kcal·(mol·mol·L~(-1))~(-1))。荧光光谱法研究固定化对DhaA去折叠过程及热力学参数的影响是深入研究DhaA稳定性的有效手段,能够为其他生物酶的稳定化机理研究提供方法指导。
The fluorescence spectroscopic characteristics of DhaA immobilized by amino-modified-mesocellular foam(MCF-NH_2) were investigated during denaturation procedures with urea and DMSO as denaturant by using F-4600 fluorescence spectrometer. The unfolding process of DhaA in denaturants was analyzed by phase diagram of fluorescence, and thermodynamic parameters were calculated with residual activity ratio of DhaA. The difference between the non-immobilized and immobilized DhaA in aspect of unfolding process and thermodynamic parameters were compared. The results showed that the catalytic activity of DhaA declined with the increasing concentration of denaturants, and the catalytic activity of immobilized DhaA could be maintained better than that of free DhaA at the same concentration of denaturant. The stabilization effect of MCF-NH_2 to DhaA was obvious before the denaturants concentration reached a critical concentration(5.5 mol·L~(-1) for urea, 7 mol·L~(-1) for DMSO). The unfolding procedure of DhaA induced by urea conformed to typical "two-state" model. The unfolding procedure of DhaA induced by DMSO conformed to "three-state" model, and the intermediate state of DhaA appeared at the DMSO concentration of 5.6 mol·L~(-1). Immobilization by MCF-NH_2 didn't change the degeneration process of DhaA, but could raise the thermodynamic parameters during the unfolding process of DhaA. When induced by urea, the ΔG(H_2O) was 8.51 kcal·mol~(-1) for free DhaA and was increased to 9.55 kcal·mol~(-1) for the immobilized one. However, the solvent-accessible surface area(m) increased from 3.69 to 4.00 kcal·(mol·mol·L~(-1))~(-1) after immobilization, which might be caused by the convenient moving of urea molecules into MCF-NH_2 with electrostatic attraction. The amino and hydroxyl groups in the channel of MCF-NH_2 could enhance DhaA rigidity through hydrogen bonding effectively reduced the effect of increasing of urea-accessible surface area, and improved the urea tolerance of DhaA. When DhaA was induced by DMSO, the ΔG(H_2O) values of process from fold state to intermediate state were always 12.12 kcal·mol~(-1) in the before and after immobilization. The solvent-accessible surface area of DhaA dropped from 3.39 to 2.30 kcal·(mol·mol·L~(-1))~(-1) after immobilization, which might be caused by the block effect of amino and hydroxyl groups in MCF-NH_(2 )which effectively inhibiting the entry of non-polar DMSO molecules. From intermediate state to unfold state, the exposure of hydrophobic amino acids in DhaA leaded to an increase of m value, but the m value of immobilized DhaA [4.40 kcal·(mol·mol·L~(-1))~(-1)] was still lower than the free DhaA [4.94 kcal·(mol·mol·L~(-1))~(-1)] due to the polar microenvironment in the channel. Studying the unfolding process and thermodynamic parameters by fluorescence spectrometer is an effective technological mean for the research on DhaA stability, which can also offer a methodological guidance for stabilization mechanism study for other enzymes.
The fluorescence spectroscopic characteristics of DhaA immobilized by amino-modified-mesocellular foam(MCF-NH_2) were investigated during denaturation procedures with urea and DMSO as denaturant by using F-4600 fluorescence spectrometer. The unfolding process of DhaA in denaturants was analyzed by phase diagram of fluorescence, and thermodynamic parameters were calculated with residual activity ratio of DhaA. The difference between the non-immobilized and immobilized DhaA in aspect of unfolding process and thermodynamic parameters were compared. The results showed that the catalytic activity of DhaA declined with the increasing concentration of denaturants, and the catalytic activity of immobilized DhaA could be maintained better than that of free DhaA at the same concentration of denaturant. The stabilization effect of MCF-NH_2 to DhaA was obvious before the denaturants concentration reached a critical concentration(5.5 mol·L~(-1) for urea, 7 mol·L~(-1) for DMSO). The unfolding procedure of DhaA induced by urea conformed to typical "two-state" model. The unfolding procedure of DhaA induced by DMSO conformed to "three-state" model, and the intermediate state of DhaA appeared at the DMSO concentration of 5.6 mol·L~(-1). Immobilization by MCF-NH_2 didn't change the degeneration process of DhaA, but could raise the thermodynamic parameters during the unfolding process of DhaA. When induced by urea, the ΔG(H_2O) was 8.51 kcal·mol~(-1) for free DhaA and was increased to 9.55 kcal·mol~(-1) for the immobilized one. However, the solvent-accessible surface area(m) increased from 3.69 to 4.00 kcal·(mol·mol·L~(-1))~(-1) after immobilization, which might be caused by the convenient moving of urea molecules into MCF-NH_2 with electrostatic attraction. The amino and hydroxyl groups in the channel of MCF-NH_2 could enhance DhaA rigidity through hydrogen bonding effectively reduced the effect of increasing of urea-accessible surface area, and improved the urea tolerance of DhaA. When DhaA was induced by DMSO, the ΔG(H_2O) values of process from fold state to intermediate state were always 12.12 kcal·mol~(-1) in the before and after immobilization. The solvent-accessible surface area of DhaA dropped from 3.39 to 2.30 kcal·(mol·mol·L~(-1))~(-1) after immobilization, which might be caused by the block effect of amino and hydroxyl groups in MCF-NH_(2 )which effectively inhibiting the entry of non-polar DMSO molecules. From intermediate state to unfold state, the exposure of hydrophobic amino acids in DhaA leaded to an increase of m value, but the m value of immobilized DhaA [4.40 kcal·(mol·mol·L~(-1))~(-1)] was still lower than the free DhaA [4.94 kcal·(mol·mol·L~(-1))~(-1)] due to the polar microenvironment in the channel. Studying the unfolding process and thermodynamic parameters by fluorescence spectrometer is an effective technological mean for the research on DhaA stability, which can also offer a methodological guidance for stabilization mechanism study for other enzymes.
引文
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