偶氮染料的生物毒性及其与生物大分子的结合作用
摘要
染料的毒性,特别是偶氮染料的致毒作用,已经引起各国政府的高度关注。如何快速评价偶氮染料对生态环境的毒性效应,减少其对环境及人类的危害,已成为急待研究的课题。染料分子可通过多种途径被人体吸收,并引起某种生物学作用。特别是在靶部位与关键性生物大分子的结合,可引起生物大分子各种结构和功能的异常,而发生毒负作用。近年来,关于污染物不良效应的研究,已经深入到以生物大分子作为污染物作用的靶位点,从而使人们对于污染的认识及其评价更为明确,也为污染物的防治提供更为科学的依据。本论文从两个层面对三种偶氮染料的毒性进行了研究。用发光细菌法评价了它们的急性毒性,并对致毒的机理进行了研究。通过三种染料与人血清白蛋白的结合机理和与DNA结合模式、亲和力大小以及对DNA碱基序列特异性识别的研究,预测其对人类潜在的危害。主要研究内容及结果如下：
1.利用发光菌法评价了三种偶氮染料的急性毒性,应用拉曼光谱技术测定了染料作用前后发光细菌体内生物大分子特征拉曼位移,并对发光菌与C.I.酸性红73接触前后特征拉曼位移的变化进行了详细指认。实验表明,在所测试的浓度范围内三种偶氮染料均有一定的毒性。其毒性从大到小的顺序是：C.I.酸性红73>C.I.酸性蓝113>C.I.活性红24。其中,C.I.酸性红73毒性最大,当浓度为100 mg/L,与发光细菌作用15分钟后,其发光拟制率为26.9%,毒性大小相当于氯化汞浓度为0.061mg/L时毒性,并随作用时间的延长,毒性增加。拉曼光谱也表明,三种偶氮染料和发光菌接触后,与其生物大分子发生了结合,或者使这些生物大分子所处的微环境发生了变化,导致了其特征拉曼峰的强度和位置发生不同程度的改变,影响到发光菌正常的生理代谢功能,表现在发光强度的降低。
2.应用分子对接技术,研究了还原黄素单核苷酸(FMNH2)与菌荧光素酶的最佳结合部位位点。完成了FMNH2和荧光素酶三个区域(即α亚基、β亚基和两个亚基的交界面处)的分子对接研究。根据FMNH2与菌荧光素酶每个区域的的最低结合能,识别了两个活性位点,一个在α亚基,命名为活性位点Ⅰ(active site-Ⅰ),这个活性位点与已报道的大量实验结果是一致的。另一个是在两个亚基交界面处,命名为活性位点Ⅱ(active site-Ⅱ)。FMNH2在这两个活性部位的最低结合能分别为：-32.4 KJ mol-1(活性位点Ⅱ)和-31.2 KJ mol-1(活性位点Ⅰ),表明两个活性位点具有近似的亲合力。活性位点Ⅱ的提出,有助于理解为什么杂二聚体的催化活性高于单个亚基的催化活性；为研究化学污染物对细菌发光抑制机理提供理论依据；也为基因突变研究和分子动力学模拟提供一些基础信息。
3.应用分子对接技术,完成了细菌催化发光反应的底物长链脂肪醛和三种染料在荧光素酶两个活性位点的分子对接模拟,计算出每种物质在两个活性位点的最低结合能。通过对比结合能的大小,分析了三种染料抑制细菌发光的分子机制。分子对接实验表明,C.I.酸性红73与底物黄素单核苷酸相比,在两个活性位点都不具备竞争优势,但是能抑制底物长链脂肪醛在两个活性位点的结合,应具有较强的细菌发光抑制效应。而C.I.酸性蓝113和C.I.活性红24在活性位点Ⅰ都具有较强的亲和力,能抑制酶与两种底物在活性位点Ⅰ的结合；但是在活性位点Ⅱ的的亲和力较差,结合能均为正值,而不能抑制它们在活性位点Ⅱ的结合,对细菌的发光抑制率应较小。这与细菌发光抑制实验得到的结果是一致的。
4.应用荧光光谱、紫外光谱、园二色谱和分子对接技术,研究了三种染料与人血清白蛋白结合的分子机理。理论和实验研究的结果表明,三种染料与蛋白质之间具有较高的亲和力,能自发结合形成配合物。疏水作用是主要的作用力类型,而氢键和静电作用对于形成稳定的染料-蛋白质复合物也起到重要作用。另外染料与白蛋质的结合还诱导蛋白质的二级结构发生了微弱变化。
分子对接的结果显示,两种酸性染料C.工.酸性红73和C.I.酸性蓝113与蛋白质最可能的结合位点在蛋白质第一结构域的ⅠB亚域；给体(Trp214)与受体(染料)之间的平均距离分别为3.06和2.82 nm,与实验得到的结果3.28 nm(C.I.酸性红73)和2.78(C.I.酸性蓝113)是非常接近的。而活性染料C.I.活性红24主要结合在蛋白质第二结构域的ⅡA亚域；Trp214与C.I.活性红24之间的平均距离为1.18 nm,而通过Forster能量转移理论求得的作用距离为3.11 nm,二者存在一定的误差。可能的原因是：(1)C.I.活性红24分子中含有较多的活性基团,包括带负电的三个磺酸基(-SO3)、两个氯原子(-Cl)和一个羟基(-OH),带正电的仲胺和一个三嗪环,这些活性基团使的该染料分子易与蛋白质的碱性或酸性氨基酸结合,而不能进入所研究的四个结合位点;(2)亚域ⅡA是C.I.活性红24最佳的结合位点,但是仍会有部分染料分子结合在其它几个亚域。通过Forster能量转移理论求出的作用距离应为C.I.活性红24的各结合位点与蛋白质色氨酸残基(Trp214)之间距离的平均值；其它原因需要做进一步研究。
研究结果表明,三种染料中,C.I.酸性红73与蛋白质具有最低的结合自由能ΔG(实验值)和最大的结合常数Kb,这与其具有最大的生物毒性(抑制细菌发光)是一致的。染料与蛋白质的结合,可能会影响到生物体内重要物质的储存和运输,与它们形成竞争,干扰正常的生理功能,导致生物毒性的发生。所以,通过研究染料与蛋白质的结合,计算它们结合力的大小和结合位点,来预测它们的生物毒性是可行的。
5.应用分子对接技术,研究了这三种染料与9个DNA片段的结合模型和对DNA序列的选择性。分子对接结果表明,小沟槽结合是三种染料与正常的DNA片段(没有插入缝隙)结合的主要结合模式。即使DNA片段中存在插入缝隙,小沟槽结合也可能是部分染料的优先结合模式。在研究的九个不同碱基序列的DNA片段中,十二聚体d(CGCGATATCGCG)2 (PDB ID:1DNE)是酸性红C.I.73最佳的结合对象,具有最低的结合自由能(-9.19kcal/mol.);六聚体d(CGATCG)2 (PDB ID:1Z3F)是C.I.酸性蓝113最佳的结合靶标,具有最低的结合自由能(-11.8kcal/mol.);含有两个插入缝隙的四聚体d(CGCG)2 (PDB ID:1D32)的小沟槽是C.I.活性红24最佳的结合靶标,具有最低的结合自由能(-8.13 kcal/mol)。结合模型分析表明,三种染料中,只有C.I.酸性红73分子中的萘二磺酸部分对DNA小沟槽的CG碱基序列具有选择性,而其它两种染料没有明显地表现出碱基序列的选择性。
研究表明,三种染料与DNA的亲合力大小跟与小沟槽结合的芳香基团的数目有关,芳环越多亲合力越大。由于C.I.酸性蓝113比其它两种染料具有更多的芳香基团与小沟槽结合,能与DNA小沟槽形成更大的π键堆积。所以除了一个十二聚体d(CGCGATATCGCG)2 (PDB ID:1DNE)外,C.I.酸性蓝113与其它8个DNA片段的结合,都具有最强的结合能力。这是否表明该种染料具有较强的基因毒性,还需要做更深入的研究。
DNA大沟槽是结合蛋白质的场所,研究发现,有部分构象的染料分子结合在DNA大沟槽中。如果染料分子与DNA大沟槽形成竞争性占位,而影响到与蛋白质的相互识别,可能会干扰生物体正常的生理功能,而产生毒性。
The toxicity of dyes, especially azo dyes, has been highly paid attention to by all governments around the world. How to rapidly evaluate the toxicity effect of azo dyes to the ecological environment in order to minimize its negative effects has become a pressing research issue. Dye molecules can be absorbed through various channels by the human body, causing certain biological effects. Especially their binding with key biological macromolecules in the target areas results in abnormal structure and function of biological macromolecules, thus causing poisonous effects. In this dissertation the toxicity of three azo dyes was studied on two levels. First, their acute toxicity was evaluated by luminous bacteria method and the mechanism of toxicity was analyzed by molecular docking technology. To predict potential dangers to human beings, we conducted research on the molecular mechanism of three kinds of azo dyes binding to human serum albumin, and on the possible mode of binding of the dyes with DNA. The main contents and results are following:
1. The acute toxicity of three azo dyes (C.I. Acid red 73, C.I. Acid blue 113 and C.I. Reactive red 24) was evaluated by bioluminescence tests. Raman shift of biological macromolecules in luminous bacteria before and after exposing dyes was measured. The results were shown that all the three kinds of azo dyes contain certain level of toxicity within the range of experiment concentration. The descending order of their toxicity is:C.I. Acid red 73> C.I. Acid blue 113> C.I. Reactive red 24. Among them, C.I. Acid red 73 has the highest toxicity. Luminescence inhibition ratio of C.I. Acid red 73 (100 mg/L, exposing 15 min) is 26.9%, whose toxicity equals to HgCl2 at the concentration level of 0.061 mg/L, and the longer the time the higher the toxicity level. Raman spectrum of the luminous bacteria also shows that the three azo dyes either combined with their biological macromolecules or changed the micro-environment of macromolecules, resulting in various changes in the strength and location of Raman peaks, which further influences the normal physiological metabolism processes of luminous bacteria reflected by their reduced luminescence strength.
2. The best binding site of flavin mononucleotide (FMNH2) and bacterial luciferase was studied applying molecular docking technology, and completed molecule docking research on FMNH2 and three areas (αsubunit,βsubunit and the interface between the two subunits) of the bacterial luciferase. Based on the lowest binding energy of FMNH2 and the three areas, two active sites were identified, one locates at a subunit, designated as active site-Ⅰ, which is consistent with the abundance of experimental results reported previously; the other locates at the interface between two subunits, designated as active site-Ⅱ. The binding energy of FMNH2 on the two sites is-32.4 KJ mol-1 (active site-Ⅱ) and -31.2 KJ mol-1 (active site-Ⅰ), indicating a similar affinity on the two sites. The introduction of active site-Ⅱhelps to understand why theα/βheterodimer has higher catalytic activity than the individualαandβsubunits, and provide theoretical basis for the study of how chemical contaminants affect luminescence strength of the bacteria as well as the studies on gene mutations and molecular dynamics simulation.
3. Molecular docking technology was applied to complete the docking simulation of four long chain aliphatic aldehydes and the three azo dyes on the two active sites of the bacterial luciferase. The lowest binding energy of each substance on these two sites was calculated. The molecular mechanism of inhibiting the luminescence strength of bacteria of three dyes was analyzed through comparing the binding energy. As is shown by the molecule docking experiment, comparing to the FMNH2, C.I. Acid red 73 is less competitive on the two sites, but it is able to inhibit the binding of and the long chain aliphatic aldehyde on the two active sites, thus possesses great potential in inhibiting luminescence. Whereas both C.I. Acid blue113 and C.I. Reactive red 24 have high affinity on active site-Ⅰand are able to prevent the binding of enzymes and the two objects on active site-Ⅰ, but have low affinity on active site-Ⅱshown by their positive value of binding energy, thus cannot inhibit their binding on active site-Ⅱand luminescence of bacteria. This is consistent with the results of luminescence experiment.
4. The molecular mechanism of the three dyes binding to human serum albumin (HSA) was investigated by fluorescence, UV-visible, far-UV CD spectroscopy and molecule docking technology. Theories and experiments have revealed high affinity ability between the three dyes and the protein to spontaneously form coordination complexes. The hydrophobic interaction is the main pattern of driving force, and the hydrogen bond and static electricity effect also play an important role in forming stable dye-protein compounds. Besides, a small change in the secondary structure of the protein was also induced by the combination of dyes and the protein.
As the results of molecule docking shows, the most possible binding site of the two acidic dyes C.I. Acid red 73 and C.I. Acid blue113 and the protein locates within the IB sub-domain of the protein. The average distances between the donor (Trp214) and the acceptor (dyes) are 3.06 and 2.82 nm respectively, which are very similar to the results from the experiment:3.28 nm (C.I. Acid red 73) and 2.78 (C.I. Acid blue 113). The main binding area of the C.I. Reactive red 24 is within the IIA sub-domain of the protein. The average distance between Trp214 and C.I Reactive red 24 is 1.18 nm, while the result obtained through Forster energy transfer theory is 3.11 nm, indicating certain level of error between the two. The reasons for this could be:(1) the molecules of C.I. Reactive red 24 are rich in active groups, including negatively charged three-SO3, two-Cl and a-OH as well as positively charged di-amine and a triazine ring. These active particles facilitate the binding of molecules to amino acid in the protein, either alkalescent or acidic, instead of binding to the four sites under this study; (2) IIA sub-domain is the best binding site for C.I. Reactive red 24, but there are also some molecules binding in other sub-domains. The distance obtained through Forster energy transfer theory should be the average of the distances between all the binding sites of C.I. Reactive red 24 and Trp214; further research needs to be done to uncover other reasons.
According to the results, among the three dyes, C.I. Acid red 73 has the lowest binding free energy△G (the results from the experiment) and highest binding constant Kb, which is consistent with the fact that it has the highest toxicity (shown by bioluminescence tests). The binding of dyes with protein may affect the storage and transportation of vital substances in the organism by forming a competition, which consequently disturbs normal physiologic function leading to the occurrence of toxicity. Therefore, through investigating the binding of dye with protein, it is possible to predict biological toxic by calculating their binding energy and binding sites.
5. Molecular docking techniques were applied to describe the most probable mode of nine DNA fragments binding as well as the sequence selectivity of the three dyes. As shown by the molecule docking experiment, the minor groove binding is the main pattern of the three dyes binding to normal DNA fragments (without intercalation gaps). The minor groove binding is the most preferable binding model of some dyes although DNA targets present intercalation gap. By analyzing the binding model, only the naphthalenedisulfonic acid moiety of the C.I. Acid red 73 selectively binds to the CG-rich region of DNA minor groove, while other dyes are not obviously selective to sequences of base pairs. Research shows that the affinity of the three dyes to DNA relates to the number of aromatic groups bound to the minor groove, and the more the aromatic rings the greater the affinity. Comparing to other two dyes, due to more aromatic rings bound to the minor groove to form a biggerπ-π-stack in C.I. Acid blue 113, apart from one dodecamer, C.I. Acid blue 113 has the highest binding energy to other eight DNA fragments. Further research needs to be done to decide whether this indicates greater genetic toxic of the dye.
The major groove of DNA is the place of combining proteins where dye molecules with some conformation combined in this study. If the dye molecules form a competition with proteins, thus influencing the recognition of proteins, the normal physiological function of organism may be disturbed, leading to the production of toxicity.
1.利用发光菌法评价了三种偶氮染料的急性毒性,应用拉曼光谱技术测定了染料作用前后发光细菌体内生物大分子特征拉曼位移,并对发光菌与C.I.酸性红73接触前后特征拉曼位移的变化进行了详细指认。实验表明,在所测试的浓度范围内三种偶氮染料均有一定的毒性。其毒性从大到小的顺序是：C.I.酸性红73>C.I.酸性蓝113>C.I.活性红24。其中,C.I.酸性红73毒性最大,当浓度为100 mg/L,与发光细菌作用15分钟后,其发光拟制率为26.9%,毒性大小相当于氯化汞浓度为0.061mg/L时毒性,并随作用时间的延长,毒性增加。拉曼光谱也表明,三种偶氮染料和发光菌接触后,与其生物大分子发生了结合,或者使这些生物大分子所处的微环境发生了变化,导致了其特征拉曼峰的强度和位置发生不同程度的改变,影响到发光菌正常的生理代谢功能,表现在发光强度的降低。
2.应用分子对接技术,研究了还原黄素单核苷酸(FMNH2)与菌荧光素酶的最佳结合部位位点。完成了FMNH2和荧光素酶三个区域(即α亚基、β亚基和两个亚基的交界面处)的分子对接研究。根据FMNH2与菌荧光素酶每个区域的的最低结合能,识别了两个活性位点,一个在α亚基,命名为活性位点Ⅰ(active site-Ⅰ),这个活性位点与已报道的大量实验结果是一致的。另一个是在两个亚基交界面处,命名为活性位点Ⅱ(active site-Ⅱ)。FMNH2在这两个活性部位的最低结合能分别为：-32.4 KJ mol-1(活性位点Ⅱ)和-31.2 KJ mol-1(活性位点Ⅰ),表明两个活性位点具有近似的亲合力。活性位点Ⅱ的提出,有助于理解为什么杂二聚体的催化活性高于单个亚基的催化活性；为研究化学污染物对细菌发光抑制机理提供理论依据；也为基因突变研究和分子动力学模拟提供一些基础信息。
3.应用分子对接技术,完成了细菌催化发光反应的底物长链脂肪醛和三种染料在荧光素酶两个活性位点的分子对接模拟,计算出每种物质在两个活性位点的最低结合能。通过对比结合能的大小,分析了三种染料抑制细菌发光的分子机制。分子对接实验表明,C.I.酸性红73与底物黄素单核苷酸相比,在两个活性位点都不具备竞争优势,但是能抑制底物长链脂肪醛在两个活性位点的结合,应具有较强的细菌发光抑制效应。而C.I.酸性蓝113和C.I.活性红24在活性位点Ⅰ都具有较强的亲和力,能抑制酶与两种底物在活性位点Ⅰ的结合；但是在活性位点Ⅱ的的亲和力较差,结合能均为正值,而不能抑制它们在活性位点Ⅱ的结合,对细菌的发光抑制率应较小。这与细菌发光抑制实验得到的结果是一致的。
4.应用荧光光谱、紫外光谱、园二色谱和分子对接技术,研究了三种染料与人血清白蛋白结合的分子机理。理论和实验研究的结果表明,三种染料与蛋白质之间具有较高的亲和力,能自发结合形成配合物。疏水作用是主要的作用力类型,而氢键和静电作用对于形成稳定的染料-蛋白质复合物也起到重要作用。另外染料与白蛋质的结合还诱导蛋白质的二级结构发生了微弱变化。
分子对接的结果显示,两种酸性染料C.工.酸性红73和C.I.酸性蓝113与蛋白质最可能的结合位点在蛋白质第一结构域的ⅠB亚域；给体(Trp214)与受体(染料)之间的平均距离分别为3.06和2.82 nm,与实验得到的结果3.28 nm(C.I.酸性红73)和2.78(C.I.酸性蓝113)是非常接近的。而活性染料C.I.活性红24主要结合在蛋白质第二结构域的ⅡA亚域；Trp214与C.I.活性红24之间的平均距离为1.18 nm,而通过Forster能量转移理论求得的作用距离为3.11 nm,二者存在一定的误差。可能的原因是：(1)C.I.活性红24分子中含有较多的活性基团,包括带负电的三个磺酸基(-SO3)、两个氯原子(-Cl)和一个羟基(-OH),带正电的仲胺和一个三嗪环,这些活性基团使的该染料分子易与蛋白质的碱性或酸性氨基酸结合,而不能进入所研究的四个结合位点;(2)亚域ⅡA是C.I.活性红24最佳的结合位点,但是仍会有部分染料分子结合在其它几个亚域。通过Forster能量转移理论求出的作用距离应为C.I.活性红24的各结合位点与蛋白质色氨酸残基(Trp214)之间距离的平均值；其它原因需要做进一步研究。
研究结果表明,三种染料中,C.I.酸性红73与蛋白质具有最低的结合自由能ΔG(实验值)和最大的结合常数Kb,这与其具有最大的生物毒性(抑制细菌发光)是一致的。染料与蛋白质的结合,可能会影响到生物体内重要物质的储存和运输,与它们形成竞争,干扰正常的生理功能,导致生物毒性的发生。所以,通过研究染料与蛋白质的结合,计算它们结合力的大小和结合位点,来预测它们的生物毒性是可行的。
5.应用分子对接技术,研究了这三种染料与9个DNA片段的结合模型和对DNA序列的选择性。分子对接结果表明,小沟槽结合是三种染料与正常的DNA片段(没有插入缝隙)结合的主要结合模式。即使DNA片段中存在插入缝隙,小沟槽结合也可能是部分染料的优先结合模式。在研究的九个不同碱基序列的DNA片段中,十二聚体d(CGCGATATCGCG)2 (PDB ID:1DNE)是酸性红C.I.73最佳的结合对象,具有最低的结合自由能(-9.19kcal/mol.);六聚体d(CGATCG)2 (PDB ID:1Z3F)是C.I.酸性蓝113最佳的结合靶标,具有最低的结合自由能(-11.8kcal/mol.);含有两个插入缝隙的四聚体d(CGCG)2 (PDB ID:1D32)的小沟槽是C.I.活性红24最佳的结合靶标,具有最低的结合自由能(-8.13 kcal/mol)。结合模型分析表明,三种染料中,只有C.I.酸性红73分子中的萘二磺酸部分对DNA小沟槽的CG碱基序列具有选择性,而其它两种染料没有明显地表现出碱基序列的选择性。
研究表明,三种染料与DNA的亲合力大小跟与小沟槽结合的芳香基团的数目有关,芳环越多亲合力越大。由于C.I.酸性蓝113比其它两种染料具有更多的芳香基团与小沟槽结合,能与DNA小沟槽形成更大的π键堆积。所以除了一个十二聚体d(CGCGATATCGCG)2 (PDB ID:1DNE)外,C.I.酸性蓝113与其它8个DNA片段的结合,都具有最强的结合能力。这是否表明该种染料具有较强的基因毒性,还需要做更深入的研究。
DNA大沟槽是结合蛋白质的场所,研究发现,有部分构象的染料分子结合在DNA大沟槽中。如果染料分子与DNA大沟槽形成竞争性占位,而影响到与蛋白质的相互识别,可能会干扰生物体正常的生理功能,而产生毒性。
The toxicity of dyes, especially azo dyes, has been highly paid attention to by all governments around the world. How to rapidly evaluate the toxicity effect of azo dyes to the ecological environment in order to minimize its negative effects has become a pressing research issue. Dye molecules can be absorbed through various channels by the human body, causing certain biological effects. Especially their binding with key biological macromolecules in the target areas results in abnormal structure and function of biological macromolecules, thus causing poisonous effects. In this dissertation the toxicity of three azo dyes was studied on two levels. First, their acute toxicity was evaluated by luminous bacteria method and the mechanism of toxicity was analyzed by molecular docking technology. To predict potential dangers to human beings, we conducted research on the molecular mechanism of three kinds of azo dyes binding to human serum albumin, and on the possible mode of binding of the dyes with DNA. The main contents and results are following:
1. The acute toxicity of three azo dyes (C.I. Acid red 73, C.I. Acid blue 113 and C.I. Reactive red 24) was evaluated by bioluminescence tests. Raman shift of biological macromolecules in luminous bacteria before and after exposing dyes was measured. The results were shown that all the three kinds of azo dyes contain certain level of toxicity within the range of experiment concentration. The descending order of their toxicity is:C.I. Acid red 73> C.I. Acid blue 113> C.I. Reactive red 24. Among them, C.I. Acid red 73 has the highest toxicity. Luminescence inhibition ratio of C.I. Acid red 73 (100 mg/L, exposing 15 min) is 26.9%, whose toxicity equals to HgCl2 at the concentration level of 0.061 mg/L, and the longer the time the higher the toxicity level. Raman spectrum of the luminous bacteria also shows that the three azo dyes either combined with their biological macromolecules or changed the micro-environment of macromolecules, resulting in various changes in the strength and location of Raman peaks, which further influences the normal physiological metabolism processes of luminous bacteria reflected by their reduced luminescence strength.
2. The best binding site of flavin mononucleotide (FMNH2) and bacterial luciferase was studied applying molecular docking technology, and completed molecule docking research on FMNH2 and three areas (αsubunit,βsubunit and the interface between the two subunits) of the bacterial luciferase. Based on the lowest binding energy of FMNH2 and the three areas, two active sites were identified, one locates at a subunit, designated as active site-Ⅰ, which is consistent with the abundance of experimental results reported previously; the other locates at the interface between two subunits, designated as active site-Ⅱ. The binding energy of FMNH2 on the two sites is-32.4 KJ mol-1 (active site-Ⅱ) and -31.2 KJ mol-1 (active site-Ⅰ), indicating a similar affinity on the two sites. The introduction of active site-Ⅱhelps to understand why theα/βheterodimer has higher catalytic activity than the individualαandβsubunits, and provide theoretical basis for the study of how chemical contaminants affect luminescence strength of the bacteria as well as the studies on gene mutations and molecular dynamics simulation.
3. Molecular docking technology was applied to complete the docking simulation of four long chain aliphatic aldehydes and the three azo dyes on the two active sites of the bacterial luciferase. The lowest binding energy of each substance on these two sites was calculated. The molecular mechanism of inhibiting the luminescence strength of bacteria of three dyes was analyzed through comparing the binding energy. As is shown by the molecule docking experiment, comparing to the FMNH2, C.I. Acid red 73 is less competitive on the two sites, but it is able to inhibit the binding of and the long chain aliphatic aldehyde on the two active sites, thus possesses great potential in inhibiting luminescence. Whereas both C.I. Acid blue113 and C.I. Reactive red 24 have high affinity on active site-Ⅰand are able to prevent the binding of enzymes and the two objects on active site-Ⅰ, but have low affinity on active site-Ⅱshown by their positive value of binding energy, thus cannot inhibit their binding on active site-Ⅱand luminescence of bacteria. This is consistent with the results of luminescence experiment.
4. The molecular mechanism of the three dyes binding to human serum albumin (HSA) was investigated by fluorescence, UV-visible, far-UV CD spectroscopy and molecule docking technology. Theories and experiments have revealed high affinity ability between the three dyes and the protein to spontaneously form coordination complexes. The hydrophobic interaction is the main pattern of driving force, and the hydrogen bond and static electricity effect also play an important role in forming stable dye-protein compounds. Besides, a small change in the secondary structure of the protein was also induced by the combination of dyes and the protein.
As the results of molecule docking shows, the most possible binding site of the two acidic dyes C.I. Acid red 73 and C.I. Acid blue113 and the protein locates within the IB sub-domain of the protein. The average distances between the donor (Trp214) and the acceptor (dyes) are 3.06 and 2.82 nm respectively, which are very similar to the results from the experiment:3.28 nm (C.I. Acid red 73) and 2.78 (C.I. Acid blue 113). The main binding area of the C.I. Reactive red 24 is within the IIA sub-domain of the protein. The average distance between Trp214 and C.I Reactive red 24 is 1.18 nm, while the result obtained through Forster energy transfer theory is 3.11 nm, indicating certain level of error between the two. The reasons for this could be:(1) the molecules of C.I. Reactive red 24 are rich in active groups, including negatively charged three-SO3, two-Cl and a-OH as well as positively charged di-amine and a triazine ring. These active particles facilitate the binding of molecules to amino acid in the protein, either alkalescent or acidic, instead of binding to the four sites under this study; (2) IIA sub-domain is the best binding site for C.I. Reactive red 24, but there are also some molecules binding in other sub-domains. The distance obtained through Forster energy transfer theory should be the average of the distances between all the binding sites of C.I. Reactive red 24 and Trp214; further research needs to be done to uncover other reasons.
According to the results, among the three dyes, C.I. Acid red 73 has the lowest binding free energy△G (the results from the experiment) and highest binding constant Kb, which is consistent with the fact that it has the highest toxicity (shown by bioluminescence tests). The binding of dyes with protein may affect the storage and transportation of vital substances in the organism by forming a competition, which consequently disturbs normal physiologic function leading to the occurrence of toxicity. Therefore, through investigating the binding of dye with protein, it is possible to predict biological toxic by calculating their binding energy and binding sites.
5. Molecular docking techniques were applied to describe the most probable mode of nine DNA fragments binding as well as the sequence selectivity of the three dyes. As shown by the molecule docking experiment, the minor groove binding is the main pattern of the three dyes binding to normal DNA fragments (without intercalation gaps). The minor groove binding is the most preferable binding model of some dyes although DNA targets present intercalation gap. By analyzing the binding model, only the naphthalenedisulfonic acid moiety of the C.I. Acid red 73 selectively binds to the CG-rich region of DNA minor groove, while other dyes are not obviously selective to sequences of base pairs. Research shows that the affinity of the three dyes to DNA relates to the number of aromatic groups bound to the minor groove, and the more the aromatic rings the greater the affinity. Comparing to other two dyes, due to more aromatic rings bound to the minor groove to form a biggerπ-π-stack in C.I. Acid blue 113, apart from one dodecamer, C.I. Acid blue 113 has the highest binding energy to other eight DNA fragments. Further research needs to be done to decide whether this indicates greater genetic toxic of the dye.
The major groove of DNA is the place of combining proteins where dye molecules with some conformation combined in this study. If the dye molecules form a competition with proteins, thus influencing the recognition of proteins, the normal physiological function of organism may be disturbed, leading to the production of toxicity.
引文
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