Adsorption Induced Enzyme Denaturation: the Role of Polymer Hydrophobicity in Adsorption and Denaturation of α-Chymotrypsin on Allyl Glycidyl Ether (AGE)-Ethylene Glycol Dimethacrylate (EGDM) Copolymers
Adsorption Induced Enzyme Denaturation: the Role of Polymer Hydrophobicity in Adsorption and Denaturation of α-Chymotrypsin on Allyl Glycidyl Ether (AGE)-Ethylene Glycol Dimethacrylate (EGDM) Copolymers
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DOI：
10.1021/la904114u
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发表时间：
2010-01-19
期刊：
影响因子：
3.9
通讯作者：
Ponrathnam, Surendra
中科院分区：
文献类型：
--
作者：
Lahari, Challa;Jasti, Lakshmi S.;Ponrathnam, Surendra
以烯丙基缩水甘油酯（AGE）和乙二醇二甲基丙烯酸酯（EGDM）为共聚物，研究了随着EGDM与AGE的摩尔比（交联密度0.05 ~ 1.5）的增加，聚合载体油的疏水性、结构和α -胰凝乳蛋白酶（e.c 3.4.21.1）活性的变化。在室温下，酶很容易在Langmuir吸附等温线（25% w/w）下吸附在水缓冲液前。通过研究该系列共聚物对萘和fmoc -蛋氨酸的吸附性能，评价了该系列共聚物的相对疏水性。当交联密度从0.05增加到0.25时，聚合物疏水性呈线性增加。交联密度的进一步增加导致萘的结合减少，但对Fmoc-Met的结合影响不大。α -凝乳胰蛋白酶与这些共聚物的结合遵循fmoc -蛋氨酸结合的趋势，而不是萘结合，表明在蛋白质与聚合物结合过程中，极性相互作用和疏水相互作用都参与其中。吸附蛋白的荧光、CID和拉曼光谱显示，吸附的酶经过广泛的变性（约80%），与共聚物接触时失去三级和二级结构。与Eupergit C、大孔Amberlite XAD-2和XAD-7的酶吸附行为比较表明，EGDM酯官能团与蛋白质的极性相互作用在酶变性中起重要作用。
Effects of changes in hydrophobicity of polymeric support oil structure and activity of alpha-chymotrypsin (E.C.3.4.21.1) have been studied with copolymers of allyl glycidyl ether (AGE) and ethylene glycol dimethacrylate (EGDM) with increasing molar ratio of EGDM to AGE (cross-link density 0.05 to 1.5). The enzyme is readily adsorbed front aqueous buffer at room temperature following Langmuir adsorption isotherms in unexpectedly large amounts (25% w/w). Relative hydrophobicity of the copolymers has been assessed by studying adsorption of naphthalene and Fmoc-methionine by the series of copolymers from aqueous solutions. Polymer hydrophobicity appears to increase linearly oil increasing cross-link density from 0.05 to 0.25. Further increase in cross-link density Causes a decrease in naphthalene binding but has little effect on binding of Fmoc-Met. Binding of alpha-chymotrypsin to these copolymers follow the trend for Fmoc-methionine binding, rather than naphthalene binding, indicating involvement of polar interactions along with hydrophobic interactions during binding of protein to the polymer. The adsorbed enzyme undergoes extensive denaturation (ca. 80%) with loss of both tertiary and secondary structure on contact with the copolymers as revealed by fluorescence, CID and Raman spectra of the adsorbed protein. Comparison of enzyme adsorption behavior with Eupergit C, macroporous Amberlite XAD-2, and XAD-7 Suggests that polar interactions of the EGDM ester functional groups with the protein play a significant role in enzyme denaturation.