溶菌酶淀粉样纤维化的分子机理
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摘要
迄今为止,已确认有20多种人类疾病与蛋白质或者多肽淀粉样纤维化有关,如Alzheimer's疾病、疯牛病、帕金森病,家族性血管淀粉样变性,溶菌酶淀粉样变性疾病等等。最近的研究发现,一些与疾病不相关的蛋白质和多肽,如Aβ肽、胰岛素、溶菌酶等,在一定的环境下,如低pH、加热、搅拌、加入助溶剂或者变性剂等,同样可以自组装形成纤维结构,表现出淀粉样纤维的特性。因此,可以说淀粉样纤维化是所有蛋白质的一个共性。而蛋白质是生物体的主要构成部分,是一切生命的物质基础,它在生物的生命活动中起着重要作用,因此对蛋白质淀粉样纤维化分子机制及其相关疾病的诊断治疗已成为目前的重要科学问题之一。
蛋白质淀粉样纤维化是一个复杂的过程,单体经自组装形成一系列大小形状不同的中间体,如寡聚体、原纤维,最终形成纤维。通常情况下,淀粉样纤维会聚集在各种器官或组织的细胞膜上,引发细胞或组织功能性障碍,从而导致相关病症的发生。目前对淀粉样纤维化的形成机理和致病机制仍不是很清楚,因此进一步的探索是十分必要的。
本文选用了鸡蛋清溶菌酶为研究对象,探索蛋白质二硫键和巯基化合物在溶菌酶淀粉样纤维化中的作用。第一部分,通过对溶菌酶二硫键进行化学标记,追踪溶菌酶分子在纤维化过程中的构象变化,如α螺旋、β-折叠、疏水区及二硫键的暴露等；第二部分,利用含巯基的小分子化合物谷胱甘肽、半胱氨酸作为抑制剂,探索蛋白质二硫键和巯基化合物在溶菌酶淀粉样纤维化过程中的作用。
实验主要方法和结果
第一部分
1.溶菌酶纤维生长的检测
采用荧光探针技术和圆二色谱检测溶菌酶在淀粉样纤维化过程中的生长和构象变化。如ThT (Thioflavin T)荧光检测β折叠、ANS (1-anilino- naphthalene 8-sulfonate)荧光检测表面疏水区改变情况。结果表明,溶菌酶在纤维化过程中主要伴随着a-螺旋减少,同时β-折叠增加,疏水区域逐渐暴露,其纤维生长曲线呈“s”型,具有成核-延伸-形成纤维的动力学特征。
2.纤维化过程中二硫键的标记及测定
通过DTT将二硫键还原为自由巯基,并分别用巯基封闭试剂NEM (N-ethylmaleimide), IAM (Iodoacetamide)对溶菌酶分子表面和内部的自由巯基进行标记,然后采用MALDI-TOF-MS分析标记序列,以此追踪溶菌酶分子在纤维化过程中二硫键的暴露次序。实验结果表明,在溶菌酶在淀粉样纤维化过程中,其64位、115位和127位半胱氨酸优先处于分子外部,6位、30位次之,76位、80位和94位在纤维成熟后期才暴露出来。对肽链的疏水性分析表明,优先暴露的半胱氨酸处于蛋白质的亲水区域,而纤维生长过程中逐渐外露的半胱氨酸则处于疏水区,与ANS荧光检测表面疏水区变化的结果相符。
第二部分
1.巯基试剂对溶菌酶纤维化的影响
采用ThT荧光检测淀粉样纤维的生长。结果表明,还原型谷胱甘肽、半胱氨酸、DTT(Dithiothreitol)对溶菌酶淀粉样纤维的形成具有抑制作用,抑制作用与浓度呈正相关。与之比较,氧化型谷胱甘肽对纤维的生长没有影响,说明巯基在抑制溶菌酶淀粉样纤维化具有关键的作用。
采用ANS荧光检测纤维疏水性的暴露情况,结果表明,还原型谷胱甘肽可以延迟溶菌酶分子的疏水区外露。
圆二色谱可用于检测蛋白质纤维化过程中二级结构变化情况,结果表明,还原型谷胱甘肽可使溶菌酶α螺旋含量明显增加；在孵育过程中,同样可观察到溶菌酶α螺旋下降、β-折叠增加的总体趋势,符合淀粉样纤维化的动力学特征。
2.纤维形态的检测
主要采用偏光显微镜和透射电子显微镜检测纤维形态。成熟的纤维在偏光显微镜下具有特征的亮黄色；而在透射电镜下,可观察到典型的丝状纤维结构。在还原型谷胱甘肽的存在下,纤维的形成受到了抑制。当还原型谷胱甘肽浓度较高时,溶菌酶不能形成典型的纤维结构。
3.溶血试验检测溶菌酶纤维的毒性
由于还原型谷胱甘肽对溶菌酶纤维化有抑制作用,并可导致形成典型纤维结构的趋势降低,所以还原型谷胱甘肽能够降低溶菌酶纤维的细胞毒性作用,并与浓度呈正相关,与ThT荧光检测的结果相符合。
4.还原型谷胱甘肽抑制溶菌酶纤维化的分子机理
电泳对比实验证实,还原型谷胱甘肽可以促进溶菌酶的水解。这种水解作用使得溶菌酶形成分子量较小的多肽,抑制了分子的自组装方式和进程。另外,巯基化合物还可能通过与溶菌酶分子的二硫键发生相互作用,从而抑制的纤维生长。
结论：溶菌酶在本文的实验条件下可以形成淀粉样纤维,在纤维化过程中,伴随着溶菌酶分子的α螺旋减少,β-折叠增加,以及疏水区域外露。利用双重化学标记-质谱进行分析,证实溶菌酶在形成淀粉样纤维过程中,分子的疏水区随着纤维化进程而逐渐外露,从而使淀粉样纤维表面疏水性增加,细胞毒性增强。含巯基的小分子化合物,如还原型谷胱甘肽、半胱氨酸和DTT对溶菌酶淀粉样纤维的形成具有抑制作用,抑制作用与浓度呈正相关。较高浓度下,溶菌酶不能形成典型的纤维结构,细胞毒性作用也随之降低。巯基化合物通过促进溶菌酶水解,以及与溶菌酶分子的二硫键发生相互作用,抑制了溶菌酶分子的自组装方式和纤维化进程。
More than 20 human diseases have been recognized to be associated with amyloid fibrils of proteins and peptides, including prion disease, Alzheimer's disease, Parkinson disease, familial vascular amyloidosis and lysozyme amyloidosis. Recent studies have demonstrated that some proteins which are not related to human diseases are also able to form amyloid fibrils under appropriate conditions; leading to a postulation that amyloid fibrillation is a common feature of all proteins and peptides. Proteins are key components in organism and play important roles in biological systems. Therefore, further elucidation of the molecular mechanism of amyloid fibrillation of proteins and development of effective methods for the treatment of amyloid diseases become challenging tasks of the scientific research.
Amyloid fibrillation of a protein is a complex process. In the duration of amyloidogenesis peptide monomers tranform and self-assembly into a cascade of intermediates with different sizes and shapes, including oligomers, profibers and mature amyloid fibers. These fibrillar aggregates deposit on cell membranes and cause organ dysfunction or disorder; leading to amyloidosis disease. The mechanism by which the fibrillar species of a protein induces cell damage has been studied extensively, although the molecular mechanism of amyloid fibrillation and the fibrillar pathogenesis is still obscure and remains further explored.
In the present work by using hen egg white lysozyme as a model protein, the roles of disulfides and sulfhydryls in the amyloid fibrillation of a protein are investigated. In PartⅠ, the conformation change of lysozyme during amyloid fibrillation was analyzed by tracking disulfide exposure and analyzingα-helix andβ-sheet elements of the protein. In PartⅡ, the inhibitory effects of sulfhydryl reagents, glutathione (GSH), cysteine and dithiothreitol (DTT), on the amyloid fibrillation of lysozyme were determined to elucidate the roles of disulfide and free sulfhydryl in amyloidogenesis of a protein.
Methods and results
PartⅠ
1. Detection of the growth of lysozyme fibrils
Fluorescent probes ThT (Thioflavin T) and ANS (1-anilino- naphthalene 8-sulfonate) have been used to monitor the growth kinetics and the surface hydrophobicity of lysozyme fibrils. The conformational change of lysozyme was analyzed by means of circular dichroism. The results showed that the hydrophobic regions of lysozyme exposed gradually during the fibril growth, accompanied by a decrease ofα-helix structure and an increase ofβ-sheet structure. The growth curve of amyloid fibrils appeared as a sigmoidal shape; indicating that the lysozyme fibrillation involved different phases including nucleation, elongation and maturation.
2. Disulfide labeling and determination
To detect the exposing order of the disulfide bonds of lysozyme in the fibrillation process, the incubated samples were treated by DTT to reduce the disulfide bonds into free sulfhydryls and labeled by blocking reagents NEM (N-ethylmaleimide) and IAM (Iodoacetamide). The resultant peptides were subjected to enzymatic digestion and MALDI-TOF-MS analysis. The results indicated that the cystein residues 64,115 and 127 were located hydrophilic surface of the protein. The next exposed were cystein residues 6 and 30, whereas cystein residues 76,80 and 94 were exposed only in the late phase of lysozyme fibrillation; suggesting these cystein residues were buried in the hydrophobic regions of the protein. These results indicated that the surfacial hydrophobicity of lysozyme is increased upon amyloid fibrillation, consistent with the data of ANS assay.
PartⅡ
1. Fibril growth in the presence of sulfhydryl reagents
ThT fluorescent was utilized to monitor the fibrillation of lysozyme in the presence of sulfhydryl reagents. The results suggested that reduced glutathione, cysteine and DTT were able to inhibit amyloid fibrillation of lysozyme in a dose-dependent manner. In contrast, the oxidized form of GSH had no effect on the lysozyme fibrillation, indicating that free sulfhydryl is a prerequisite for these compounds to inhibit amyloid fibrillation.
ANS assay demonstrated that GSH also inhibited the exposure of the buried hydrophobic region of lysozyme.
Circular dichroism is an effective tool to detect the secondary structure of proteins. The results showed that GSH increased the a-helix content of native lysozyme. Upon incubation with GSH, lysozyme transformed into amyloid morphology with a decrease of a-helix and an increase ofβ-sheet structure.
2. The fibrillar morphology
The fibrillar morphology was monitored by polarizing microscopy and transmission electron microscopy. Mature fibrils showed bright yellow under polarizing microscopy. Fibrillar filaments were observed by Transmission electron microscopy. In the presence of GSH, fibrillation of lysozyme was inhibited and amorphous aggregates were observed in the samples that high concentration of GSH was applied.
3. The effect of GSH on the cytotoxicity of lysozyme amyloid fibrils
Human erythrocytes were used as an in vitro model to determine the disruptive effect of lysozyme fibrils on cell membranes. Lysozyme fibrils were able to induce hemolysis of erythrocytes. GSH inhibited the fibrillation of lysozyme and consequently attenuated the fibrillar cytotoxicity of the protein. The inhibitory effect of GSH was dose-dependent, consistent with the results of ThT assay.
4. The molecular mechanism of the roles of GSH
SDS-PAGE results demonstrated that GSH increased the formation of hydrolytic products of lysozyme upon incubation. Hydrolysis resulted in degrading lysozyme into small peptides and consequently the fibrillation was inhibited. Moreover, lysozyme fibrillation can also be inhibited by the interactions between disulfide and free sulfhydryl groups.
Conclusion
Lysozyme can form amyloid fibrils under the conditions of the present work. Amyloid fibrillation of lysozyme was associated with exposure of the buried hydrophobic regions and changes of the secondary structures, as demonstrated by disulfide-labeling-MS analysis, circular dichroism, ThT and ANS assays. The increase in surface hydrophobicity of lysozyme assemblies resulted in an increase in cytotoxicity. Sulfhydryl reagents, reduced glutathione, cysteine and DTT inhibited amyloid fibrillation of lysozyme in a dose-dependent manner. High concentration of GSH inhibited the amyloid fibrillation, altered the fibrillar morphology and attenuated the fibrillar cytotoxicity of lysozyme. The molecular mechanism involved possibly that GSH enhanced hydrolytic rate of lysozyme and therefore the fibrillation was altered.
蛋白质淀粉样纤维化是一个复杂的过程,单体经自组装形成一系列大小形状不同的中间体,如寡聚体、原纤维,最终形成纤维。通常情况下,淀粉样纤维会聚集在各种器官或组织的细胞膜上,引发细胞或组织功能性障碍,从而导致相关病症的发生。目前对淀粉样纤维化的形成机理和致病机制仍不是很清楚,因此进一步的探索是十分必要的。
本文选用了鸡蛋清溶菌酶为研究对象,探索蛋白质二硫键和巯基化合物在溶菌酶淀粉样纤维化中的作用。第一部分,通过对溶菌酶二硫键进行化学标记,追踪溶菌酶分子在纤维化过程中的构象变化,如α螺旋、β-折叠、疏水区及二硫键的暴露等；第二部分,利用含巯基的小分子化合物谷胱甘肽、半胱氨酸作为抑制剂,探索蛋白质二硫键和巯基化合物在溶菌酶淀粉样纤维化过程中的作用。
实验主要方法和结果
第一部分
1.溶菌酶纤维生长的检测
采用荧光探针技术和圆二色谱检测溶菌酶在淀粉样纤维化过程中的生长和构象变化。如ThT (Thioflavin T)荧光检测β折叠、ANS (1-anilino- naphthalene 8-sulfonate)荧光检测表面疏水区改变情况。结果表明,溶菌酶在纤维化过程中主要伴随着a-螺旋减少,同时β-折叠增加,疏水区域逐渐暴露,其纤维生长曲线呈“s”型,具有成核-延伸-形成纤维的动力学特征。
2.纤维化过程中二硫键的标记及测定
通过DTT将二硫键还原为自由巯基,并分别用巯基封闭试剂NEM (N-ethylmaleimide), IAM (Iodoacetamide)对溶菌酶分子表面和内部的自由巯基进行标记,然后采用MALDI-TOF-MS分析标记序列,以此追踪溶菌酶分子在纤维化过程中二硫键的暴露次序。实验结果表明,在溶菌酶在淀粉样纤维化过程中,其64位、115位和127位半胱氨酸优先处于分子外部,6位、30位次之,76位、80位和94位在纤维成熟后期才暴露出来。对肽链的疏水性分析表明,优先暴露的半胱氨酸处于蛋白质的亲水区域,而纤维生长过程中逐渐外露的半胱氨酸则处于疏水区,与ANS荧光检测表面疏水区变化的结果相符。
第二部分
1.巯基试剂对溶菌酶纤维化的影响
采用ThT荧光检测淀粉样纤维的生长。结果表明,还原型谷胱甘肽、半胱氨酸、DTT(Dithiothreitol)对溶菌酶淀粉样纤维的形成具有抑制作用,抑制作用与浓度呈正相关。与之比较,氧化型谷胱甘肽对纤维的生长没有影响,说明巯基在抑制溶菌酶淀粉样纤维化具有关键的作用。
采用ANS荧光检测纤维疏水性的暴露情况,结果表明,还原型谷胱甘肽可以延迟溶菌酶分子的疏水区外露。
圆二色谱可用于检测蛋白质纤维化过程中二级结构变化情况,结果表明,还原型谷胱甘肽可使溶菌酶α螺旋含量明显增加；在孵育过程中,同样可观察到溶菌酶α螺旋下降、β-折叠增加的总体趋势,符合淀粉样纤维化的动力学特征。
2.纤维形态的检测
主要采用偏光显微镜和透射电子显微镜检测纤维形态。成熟的纤维在偏光显微镜下具有特征的亮黄色；而在透射电镜下,可观察到典型的丝状纤维结构。在还原型谷胱甘肽的存在下,纤维的形成受到了抑制。当还原型谷胱甘肽浓度较高时,溶菌酶不能形成典型的纤维结构。
3.溶血试验检测溶菌酶纤维的毒性
由于还原型谷胱甘肽对溶菌酶纤维化有抑制作用,并可导致形成典型纤维结构的趋势降低,所以还原型谷胱甘肽能够降低溶菌酶纤维的细胞毒性作用,并与浓度呈正相关,与ThT荧光检测的结果相符合。
4.还原型谷胱甘肽抑制溶菌酶纤维化的分子机理
电泳对比实验证实,还原型谷胱甘肽可以促进溶菌酶的水解。这种水解作用使得溶菌酶形成分子量较小的多肽,抑制了分子的自组装方式和进程。另外,巯基化合物还可能通过与溶菌酶分子的二硫键发生相互作用,从而抑制的纤维生长。
结论：溶菌酶在本文的实验条件下可以形成淀粉样纤维,在纤维化过程中,伴随着溶菌酶分子的α螺旋减少,β-折叠增加,以及疏水区域外露。利用双重化学标记-质谱进行分析,证实溶菌酶在形成淀粉样纤维过程中,分子的疏水区随着纤维化进程而逐渐外露,从而使淀粉样纤维表面疏水性增加,细胞毒性增强。含巯基的小分子化合物,如还原型谷胱甘肽、半胱氨酸和DTT对溶菌酶淀粉样纤维的形成具有抑制作用,抑制作用与浓度呈正相关。较高浓度下,溶菌酶不能形成典型的纤维结构,细胞毒性作用也随之降低。巯基化合物通过促进溶菌酶水解,以及与溶菌酶分子的二硫键发生相互作用,抑制了溶菌酶分子的自组装方式和纤维化进程。
More than 20 human diseases have been recognized to be associated with amyloid fibrils of proteins and peptides, including prion disease, Alzheimer's disease, Parkinson disease, familial vascular amyloidosis and lysozyme amyloidosis. Recent studies have demonstrated that some proteins which are not related to human diseases are also able to form amyloid fibrils under appropriate conditions; leading to a postulation that amyloid fibrillation is a common feature of all proteins and peptides. Proteins are key components in organism and play important roles in biological systems. Therefore, further elucidation of the molecular mechanism of amyloid fibrillation of proteins and development of effective methods for the treatment of amyloid diseases become challenging tasks of the scientific research.
Amyloid fibrillation of a protein is a complex process. In the duration of amyloidogenesis peptide monomers tranform and self-assembly into a cascade of intermediates with different sizes and shapes, including oligomers, profibers and mature amyloid fibers. These fibrillar aggregates deposit on cell membranes and cause organ dysfunction or disorder; leading to amyloidosis disease. The mechanism by which the fibrillar species of a protein induces cell damage has been studied extensively, although the molecular mechanism of amyloid fibrillation and the fibrillar pathogenesis is still obscure and remains further explored.
In the present work by using hen egg white lysozyme as a model protein, the roles of disulfides and sulfhydryls in the amyloid fibrillation of a protein are investigated. In PartⅠ, the conformation change of lysozyme during amyloid fibrillation was analyzed by tracking disulfide exposure and analyzingα-helix andβ-sheet elements of the protein. In PartⅡ, the inhibitory effects of sulfhydryl reagents, glutathione (GSH), cysteine and dithiothreitol (DTT), on the amyloid fibrillation of lysozyme were determined to elucidate the roles of disulfide and free sulfhydryl in amyloidogenesis of a protein.
Methods and results
PartⅠ
1. Detection of the growth of lysozyme fibrils
Fluorescent probes ThT (Thioflavin T) and ANS (1-anilino- naphthalene 8-sulfonate) have been used to monitor the growth kinetics and the surface hydrophobicity of lysozyme fibrils. The conformational change of lysozyme was analyzed by means of circular dichroism. The results showed that the hydrophobic regions of lysozyme exposed gradually during the fibril growth, accompanied by a decrease ofα-helix structure and an increase ofβ-sheet structure. The growth curve of amyloid fibrils appeared as a sigmoidal shape; indicating that the lysozyme fibrillation involved different phases including nucleation, elongation and maturation.
2. Disulfide labeling and determination
To detect the exposing order of the disulfide bonds of lysozyme in the fibrillation process, the incubated samples were treated by DTT to reduce the disulfide bonds into free sulfhydryls and labeled by blocking reagents NEM (N-ethylmaleimide) and IAM (Iodoacetamide). The resultant peptides were subjected to enzymatic digestion and MALDI-TOF-MS analysis. The results indicated that the cystein residues 64,115 and 127 were located hydrophilic surface of the protein. The next exposed were cystein residues 6 and 30, whereas cystein residues 76,80 and 94 were exposed only in the late phase of lysozyme fibrillation; suggesting these cystein residues were buried in the hydrophobic regions of the protein. These results indicated that the surfacial hydrophobicity of lysozyme is increased upon amyloid fibrillation, consistent with the data of ANS assay.
PartⅡ
1. Fibril growth in the presence of sulfhydryl reagents
ThT fluorescent was utilized to monitor the fibrillation of lysozyme in the presence of sulfhydryl reagents. The results suggested that reduced glutathione, cysteine and DTT were able to inhibit amyloid fibrillation of lysozyme in a dose-dependent manner. In contrast, the oxidized form of GSH had no effect on the lysozyme fibrillation, indicating that free sulfhydryl is a prerequisite for these compounds to inhibit amyloid fibrillation.
ANS assay demonstrated that GSH also inhibited the exposure of the buried hydrophobic region of lysozyme.
Circular dichroism is an effective tool to detect the secondary structure of proteins. The results showed that GSH increased the a-helix content of native lysozyme. Upon incubation with GSH, lysozyme transformed into amyloid morphology with a decrease of a-helix and an increase ofβ-sheet structure.
2. The fibrillar morphology
The fibrillar morphology was monitored by polarizing microscopy and transmission electron microscopy. Mature fibrils showed bright yellow under polarizing microscopy. Fibrillar filaments were observed by Transmission electron microscopy. In the presence of GSH, fibrillation of lysozyme was inhibited and amorphous aggregates were observed in the samples that high concentration of GSH was applied.
3. The effect of GSH on the cytotoxicity of lysozyme amyloid fibrils
Human erythrocytes were used as an in vitro model to determine the disruptive effect of lysozyme fibrils on cell membranes. Lysozyme fibrils were able to induce hemolysis of erythrocytes. GSH inhibited the fibrillation of lysozyme and consequently attenuated the fibrillar cytotoxicity of the protein. The inhibitory effect of GSH was dose-dependent, consistent with the results of ThT assay.
4. The molecular mechanism of the roles of GSH
SDS-PAGE results demonstrated that GSH increased the formation of hydrolytic products of lysozyme upon incubation. Hydrolysis resulted in degrading lysozyme into small peptides and consequently the fibrillation was inhibited. Moreover, lysozyme fibrillation can also be inhibited by the interactions between disulfide and free sulfhydryl groups.
Conclusion
Lysozyme can form amyloid fibrils under the conditions of the present work. Amyloid fibrillation of lysozyme was associated with exposure of the buried hydrophobic regions and changes of the secondary structures, as demonstrated by disulfide-labeling-MS analysis, circular dichroism, ThT and ANS assays. The increase in surface hydrophobicity of lysozyme assemblies resulted in an increase in cytotoxicity. Sulfhydryl reagents, reduced glutathione, cysteine and DTT inhibited amyloid fibrillation of lysozyme in a dose-dependent manner. High concentration of GSH inhibited the amyloid fibrillation, altered the fibrillar morphology and attenuated the fibrillar cytotoxicity of lysozyme. The molecular mechanism involved possibly that GSH enhanced hydrolytic rate of lysozyme and therefore the fibrillation was altered.
引文
[1]C.Louise. M.S.Serpell, D.Merrill, G. A.Benson, M. B.Tennent, Pepys and E. F. Paul. The Protofilament Substructure of Amyloid Fibrils[J]. Mol. Biol.2000,300: 1033-1039.
[2]C.A. Ross, M.A. Poirier, Protein aggregation and neurodegenerative disease[J], Nat.Med.2004,10:10-17.
[3]R.M. Murphy, Peptide aggregation in neurodegenerative disease[J], Annu. Rev. Biomed. Eng.2002,4:155-174.
[4]V.N. Uversky, A.L. Fink, Conformational constraints for amyloid fibrillation:the importance of being unfolded[J], Biochim. Biophys. Acta.2004,1698:131-153.
[5]M.A.Chad, A.Mikhail, Karymov, K. A. Y. Lushnikov, M.V. Andrew,N. Uversky and L. L. Yuri. Protein Interactions and Misfolding Analyzed by AFM Force Spectroscopy[J].Mol. Biol.2005,354:1028-1042.
[6]C. M.Dobson, V.Bellotti, P.Mangione & M.Stoppini.Protein misfolding, evolution and disease[J]. Trends Biochem. Sci.1999,24:329-332.
[7]T. Pettersson, MD, and Y. T. Konttinen, MD.Amyloidosis—Recent Developments [J]. Semin Arthritis Rheum 2010,39:356-368.
[8]D.Jean, A.S Sipe.Review: History of the Amyloid Fibril[J]. Journal of Structural Biology,2000,130:88-98.
[9]P.Riccardo, G.Enrico and C.Amedeo.Pathways and Intermediates of Amyloid Fibril Formation[J].Mol. Biol.2007,374:917-924.
[10]C.M. Dobson, Principles of protein folding, misfolding and aggregation[J], Semin. Cell.Dev. Biol.2004,15:3-16.
[11]S.S.S.Wang, T.A. Good, An overview of Alzheimer's disease[J]. Chin. Ins. Chem. Eng.2005,36:533-559.
[12]C.A. Ross, M.A. Poirier, Protein aggregation and neurodegenerative disease[J], Nat.Med.2004,10:10-17.
[13]V.N. Uversky, A.L. Fink, Conformational constraints for amyloid fibrillation:the importance of being unfolded, Biochim. Biophys. Acta.2004,1698:131-153.
[14]E.L.Annette, V.Bente.Methods for structural characterization of prefibrillar inter-mediates and amyloid fibrils[J].FEBS Letters,2009,583:2600-2609.
[15]C. Louise, L.Serpell, S. Margaret, D. Merrill, G.A.Benson, M. B.Tennent, Pepys and E. F. Paul. The Protofilament Substructure of Amyloid Fibrils[J]. J. Mol. Biol.2000,300:1033-1039.
[16]D.L. Rymer, T.A. Good, The role of G protein activation in the toxicity of amyloidogenic Abeta-(1-40), Abeta-(25-35), and bovine calcitonin, J. Biol. Chem.2001,276:2523-2530.
[17]D.M. Walsh, A. Lomakin, G.B. Benedek, M.M. Condron, D.B. Teplow, Amyloid beta-protein fibrillogenesis. Detection of a protofibrillar intermediate[J]. Biol. Chem.1997,272:22364-22372.
[18]R.V.Ward, K.H. Jennings, R. Jepras,W. Neville, D.E. Owen, J. Hawkins, G. Christie, J.B. Davis, A. George, E.H. Karran, D.R. Howlett, Fractionation and characterization of oligomeric, protofibrillar and fibrillar forms of beta-amyloid peptide[J], Biochem.2000,348:137-144.
[19]J.I.Guijarro, M.Sunde, J.A Jones, I.D.Campbell and C.M.Dobson.Amloid fibril formation by an SH3 domain[J], Proc.Natl.Acal.Sci.U.S.A.1998.95:4224-4228.
[20]J.Brange, L.Andrsen, E.D.Laursen,G..Meyn and E.Rasmussen.Toward under-standing insulin fibrillation[J].Pharm.Sci.1997.86:571-525.
[21]C.E. Macphee,and C.M.Dobson.Chemical dissenction and reassembly of amloid fibril formed fragment of transthyretin[J].Mol.BIOL.2000,297:1203-1215.
[22]S.Ohnishi, A.Koide and S.Koide.Solution conformation and amyliod-like fibril formation of a polar peptide detived from a a-hairpin in the OspA single-layeer a-sheet[J].Mol.BIOL.2000.301:477-489.
[23]P.T. Lansbury Jr. Evolution of amyloid:what normal protein folding may tell us about fibrillogenesis and disease[J], Proc. Natl. Acad. Sci. USA 1999,96:3 342-3344.
[24]S.R. Ji, Y. Wu, S.F. Sui, Study of the correlation of secondary structure of betaamyloid peptide (Abeta40) with the hydrophobic exposure under different conditions[J], Gen. Physiol. Biophys.2002,21:415-427.
[25]C.Soto,Alzheimer's andpriondisease asdisorders of proteinconformation: implications for the design of novel therapeutic approaches [J]. Mol. Med.1999, 77:412-418.
[26]K.Hun Mok,Jenny Pettersson,Sten Orrenius,Catharina Svanborg. HAMLET, protein folding, and tumor cell death[J]. Biochemical and Biophysical Research Communications,2007,354:1-7.
[27]K.J.Pettersson, S. Aits, L.Gustafsson et al. Can misfolded proteins be beneficial? The HAMLET case [J]. Annals of Medicine,2009,41:162-176.
[28]N.U.Vladimir.Amyloidogenesis of Natively Unfolded Proteins [J].Curr Alzheimer Res.,2008,June 5(3):260-287.
[29]J.L.Helen and N.H.Philip.Systemic amyloidosis[J].Current Opinion in Pharmaco-logy,2006,6:214-220.
[30]A.L.Leheninger, D.L. Nelson, M.M.Cox. Principles of Biochemistry. [J]. New York:Worth Publishers.1933:180,312.
[31]Lysozyme.[EB/OL].http://lysozyme.co.uk/.
[32]E.Johan, P.Joubert. The use of lysozyme[EB/OL]. http://www.wynboer.co.za/ recentarticles/0201lyso.php3
[33]J. Y.Sun, U.Chibuike, E. A.Rotimi, J.P.Wu.Multifunctional peptides from egg white lysozyme[J].Food Research International 2010,43:848-855.
[34]R.Sarma,R.Bott.Crystallographic study of turkey egg white lysozyme and its complex with a disaccharide.J Mol Biol.1997,113:555.
[35]S.S.S.Wang, S.W. Chou, K.N.Liu,C.H.Wu.Effects of glutathione on amyloid fib-rillation of hen egg-white lysozyme[J]. International Journal of Biological Macromolecules,2009,45:321-329.
[36]M.C. Vaney, S.Maignan, M. Ries-Kautt, A.Ducriux, High-resolution structure (1.33 A)of a HEW lysozyme tetragonal crystal grown in the APCF apparatus. [J].D Biol. Crystallogr.1996,52:505-517.
[37]刘莹,孙荣丹,杨翔华,王丽.溶菌酶的应用现状.《饲料工业》·2006年第27卷第6期.
[38]F.Erica, P. P. Laureto, J. Zurdo, M. D.Christopher and F .Angelo. A Highly Amyloidogenic Region of Hen Lysozyme[J].Mol. Biol.2004,340:1153-1165
[39]Bfxqt.Glutathione[EB/OL]. http://www.doc88.com/p-99827123190.html 2010,11,19
[40]D. R. Francesco, et al. Ef ficacy an d safety of high-dose cisplatin an dcyclophosphamide with glut athione prot ection in the treatment of bulky advanced epithelial ovarian cancer[J]. Cancer Chemother Pharm acol,1990, 25:355-360.
[41]Y.Z.Fang, S.Yang, G.Wu, etal. Flee radicals, antioxidants,and nutrition[J].Nutrit ion,2002,18:872-879.
[42]M.G. Niroshini., I.G.Gregory and J.Claus.Multiple roles of cysteine in bio-catalysis[J].Biochemical and Biophysical Research Communications.2003, 300:1-4.
[43]张苏,王凤山.固定化原生质体转化合成L—半胱氨酸的研究.山东大学,学位论文.
[44]F.D.Gillin,L.S.Diamond.Attaclllnent of Entamoeba histolytica to glass in a defmed Maintenance medium:specific requirement for cysteine and ascorbic acid[J].Journal of Protozoology,1980,27(4):474-478
[45]M. R Nilsson.Techniques to study amyloid fibril formation in vitro[J]. Methods, 2004,34:151-160
[46]M.Biancalana,K.Makabe,A.Koide,S.Molecular mechanism of Thioflavin-T binding to the surface of β-richpeptide self-assemblies[J].Journal of Molecular Biology,2009.385(4):1052-1063.
[47]A.L. Fink.ANS.In T.E.Creighton,editor.The Encyclopedia of monomeric insulin [J].The Journal of Biology.1999.
[48]N.Berova,K.Nakanishi,R.W.Woody(Eds.).Circular Dichroism:Principles and Applications[M].R.New York:Wiley-VCH,20,877-879.
[49]D.H.A.Corre,C.H.I.Ramos.The use of circular dichroism spectroscopy to study protein folding, form and function[J]. African Journal of Biochemistry Research,2009,3(5):164-173.
[50]N.Sreerama& R.W.Woody.A self-consistent method for the analysis of protein secondary structure from circular dichroism[J].Anal.Biochem.1993,209:32-44.
[51]S.Hermann.Tricine-SDS-PAGE.Nature Protocols[J].2006.1(1):16-22.
[52]F.P.Marcelo and L.N.Claudia.Electrophoretic Analysis (Tricine-SDS-PAGE) [J].Acta Farm.Bonaerense.2002,21(1):57-60
[53]S.Raymond,L.Weintraub.Acrylamide gel as a supporting medium for zone electrophoresis. Science[J].1959,130:711.
[54]A.L.Shapiro,E.Vinuela, J.J.Maizel. Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels[J]. Biochem Biophys Res Commun.1967,28(5):815-820.
[55]H.L.Kristian, B.H.Mevik, E.O. Rukke, T.Almφy.Quantitative whole spectrum analysis with MALDI-TOF MS, Part Ⅰ:Measurement optimization[J]. Chemometrics and Intelligent Laboratory Systems.2009.96:210-218.
[56]F.Erica, P.L.Patrizia,Z.Jesus, M.D.Christopher and F.AAngelo.Highly Amyloid-ogenic Region of Hen Lysozyme[J]. J.Mol. Biol.2004,340:1153-1165
[57]L.N.Arnaudov, V.R.De. Thermally induced fibrillar aggregation of hen egg white lysozyme[J]. Biophysical Journal,2005,88:515-526.
[58]H.Akihito,A.Hiroyuki, and K.Akio.Amyloid Fibril Formation of Hen Lysozyme Depends on the Instability of the C-Helix (88-99)[J]. Biosci. Biotechnol. Biochem.,2008,72 (6):1523-1530.
[59]H. LeVine Ⅲ, Thioflavine T interaction with synthetic Alzheimer's disease beta-amyloid peptides:detection of amyloid aggregation in solution[J],.Protein. Sci. 1993,2:404-410.
[60]H.Daniel,A.Correaland H.Carlos,I.Ramos.The use of circular dichroism spectr-oscopy to study protein folding, form and function[J]. African Journal of Biochemistry Research,2009,3(5):164-173.
[61]M.R.Nilsson.Techniques to study amyloid fibril formation in vitro[J]. Methods, 2004,34:151-160.
[62]K.Young, O.H.Sam, R.G.Natalie,Y.Korlann, V. L.Elizabeth., R.C.Frank and W. Shimon.Efficient Site-Specific Labeling of Proteins via Cysteines[J]. Biocon-jugate Chem.2008,19:786-791.
[63]R.Vassilios,H.Rasmus, C.Lydia, R.E.Stephen.Separation and identification of hen egg protein isoforms using SDS-PAGE and 2D gel electrophoresis with MALDI-TOF mass spectrometry[J]. Food Chemistry 2006,99:702-710
[64]K.Sadamu, K.Tamie, K.Naoka, N.Satomi and M.Hiroyuki. Quantitative proteome analysis using D-labeled N-ethylmaleimide and 13C-labeled iodoacetanilide by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry[J]. Bioorganic & Medicinal Chemistry.2006,14:8197-8209.
[65]M. Monti, A. Amoresano, S. Giorgetti, V. Bellotti, P. Pucci. Limited proteolysis in the investigation of h2-microglobulin amyloidogenic and fibrillar states[J]. Biochimica et Biophysica Acta 2005,1753:44-50.
[66]R.Gramer,M.Saxton,K.Barnouin. Sample preparation of gel electrophoretically separated protein binding partners for analyais by volatile solubilizing agents[J]. Methods Mol Biol.24,261:499-510.
[67]王旭初,范鹏翔,李银心,等.一种适合与质谱分析的简化叫内酶解方法[J].植物生物与分子生物学学报,2007,33(5)：449-455.
[68]W.John & L.Sons,.Proteins structure and function[M].2005.
[69]A.L. Smoot, M. Panda, B.T. Brazil, A.M. Buckle, A.R. Fersht, P.M. Horowitz, The binding of bis-ANS to the isolated GroEL apical domain fragment induces the formation of a folding intermediate with increased hydrophobic surface not observed in tetradecameric GroEL[J], Biochemistry.2001,40:4484-4492.
[70]C.P. Liu, Z.Y. Li, G.C. Huang, S. Perrett, J.M. Zhou, Two distinct intermediates of trigger factor are populated during guanidine denaturation[J], Biochimie.2005, 87:1023-1031.
[71]I.Sirangelo,E.Bismuto,S.Tavassi,G..Irace.Apomyoglobin folding intermediates characterized by the hydrophobic fluorescent probe 8-anilino-1-naphthalene sulfonate[J], Biochim. Biophys. Acta.1998,1385:69-77.
[72]S.S.S.Wang,K.N.Liu,W.H.Lee. Effect of curcumin on the amyloid fibrillogenesis of hen egg-white lysozyme[J]. Biophysical Chemistry.2009,144:78-87.
[73]M. Dumoulin, J.R. Kumita, C.M. Dobson, Normal and aberrant biological self-assembly:insights from studies of human lysozyme and its amyloidogenic variants[J], Acc. Chem. Res.2006,39:603-610.
[74]M.B. Pepys, P.N. Hawkins, D.R. Booth, D.M. Vigushin, GA. Tennent, A.K. Soutar, N.Totty, O. Nguyen, C.C. Blake, C.J. Terry, et al., Human lysozyme gene mutations cause hereditary systemic amyloidosis[J], Nature.1993,362:553-557.
[75]I.A. Cotgreave, R.G. Gerdes, Glutathione-protein interactions:A molecular link between oxidative stress and cell proliferation? [J] Bio-chem. Biophys. Res. Commun.1998,242:1-9.
[76]F. Aslund, J. Beckwith.Bridge over troubled waters:Sensing stress by disulWde bond formation[J]. Cell.1999,96:751-753.
[77]M.S. Paget, M.J. Buttner, Thiol-based regulatory switches, Annu. Rev.Genet. 2003,37:91-121.
[78]K.R.Lynette, L.L.Barbara,V.S.Charles.Detection of reversible protein thiol modi Wcations in tissues[J]. Analytical Biochemistry.2006,358:171-184.
[79]B.Huang, J.He, J.Ren, X.Y.Yan, and C.M.Zeng. Cellular Membrane Disruption by Amyloid Fibrils Involved Intermolecular Disulfide Cross-Linking[J]. Biochemistry 2009,48:5794-5800.
[80]Y. Porat, A. Abramowitz, E. Gazit, Inhibition of amyloid fibril formation by polyphenols:structural similarity and aromatic interactions as a common inhibition mechanism[J], Chem. Biol. Drug. Des.2006,67:27-37.
[81]D.R. Howlett, A.E. Perry, F. Godfrey, J.E. Swatton, K.H. Jennings, C. Spitzfaden, H.Wadsworth, S.J.Wood, R.E.Markwell, Inhibition of fibril formation in beta-amyloid peptide by a novel series of benzofurans[J], Biochem.J.1999,340:283-289.
[82]W.E. Klunk, M.L. Debnath, A.M. Koros, J.W. Pettegrew, Chrysamine-G, a lipophilic analogue of Congo red, inhibits A beta-induced toxicity in PC 12 cells[J], Life Sci.1998,63:1807-1814.
[83]T. Tomiyama, A. Shoji, K. Kataoka, Y. Suwa, S. Asano, H. Kaneko, N. Endo, Inhibition of amyloid beta protein aggregation and neurotoxicity by rifampicin. Its possible function as a hydroxyl radical scavenger[J],Biol.Chem.1996,271 6839-6844.
[84]T. Tomiyama, H. Kaneko, K. Kataoka, S. Asano, N. Endo, Rifampicin inhibits the toxicity of pre-aggregated amyloid peptides by binding to peptide fibrils and preventing amyloid-cell interaction[J], Biochem. J.1997,322:859-865.
[85]A. Lomakin, D.S. Chung, G.B. Benedek, D.A. Kirschner, D.B. Teplow, On the nucleation and growth of amyloid beta-protein fibrils:detection of nuclei and quantitation of rate constants[J], Proc. Natl. Acad. Sci. U. S. A.1996,93: 1125-1129.
[86]S.J. Wood, L. MacKenzie, B. Maleeff, M.R. Hurle, R.Wetzel, Selective inhibition of Abeta fibril formation[J], J. Biol. Chem.1996,271:4086-4092.
[87]K. Ono, Y. Yoshiike, A. Takashima, K. Hasegawa, H. Naiki, M. Yamada, Potent anti-amyloidogenic and fibril-destabilizing effects of polyphenols in vitro: implications for the prevention and therapeutics of Alzheimer's disease[J]. Neurochem.2003,87:172-181.
[88]L.D. Estrada, C. Soto, Disrupting beta-amyloid aggregation for Alzheimer disease treatment, Curr. Top[J]. Med. Chem.2007,7:115-126.
[89]R. Liu,H.Barkhordarian,S.Emadi, C.B. Park, M.R. Sierks,Trehalose differentially inhibits aggregation and neurotoxicity of beta-amyloid[J].Neurobiol.2005,20 :74-81.
[90]F.G. De Felice,M.N. Vieira, L.M. Saraiva, J.D. Figueroa-Villar, J. Garcia-Abreu, R. Liu, L.Chang, W.L. Klein, S.T. Ferreira, Targeting the neurotoxic species in Alzheimer's disease:inhibitors of Abeta oligomerization[J], Faseb J.2004,18: 1366-1372.
[91]P. N.Dekhuijzen. Antioxidant properties of Nacetylcysteine:their relevance in relation to chronic obstructive pulmonary disease[J]. Eur. Respir. J.2004,23:629-636.
[92]Y.Ohhashi, Y.Hagihara, G.Kozhukh, M.Hoshino, K.Hasegawa, I.Yamaguchi, et al. The intrachain disulfide bond of β2-microglobulin is not essential for the immunoglobulin fold at neutral pH,but is essential for amyloid fibril formation at acidic pH[J]. Biochem.2002,131:45-52.
[93]D. P.Hong, M Gozu, K.Hasegawa, H.Naiki& Y. Goto.Conformation of β2-microglobulin amyloid fibrils analyzed by reduction of the disulfide bond[J]. Biol. Chem.2002,277:21554-21560.
[94]K. Ono, K. Hasegawa, H. Naiki, M. Yamada, Curcumin has potent anti-amyloidogenic effects for Alzheimer's beta-amyloid fibrils in vitro[J]. Neurosci. Res.2004,75:742-750.
[95]K. Ono, M. Yamada, Antioxidant compounds have potent anti-fibrillogenic and fibril-destabilizing effects for alpha-synuclein fibrils in vitro[J]. Neurochem. 2006,97:105-115.
[96]F. Yang, G.P. Lim, A.N. Begum, O.J. Ubeda,M.R. Simmons, S.S. Ambegaokar, P.P. Chen,R. Kayed, C.G. Glabe, S.A. Frautschy, G.M. Cole, Curcumin inhibits formation of amyloid beta oligomers and fibrils, binds plaques, and reduces amyloid in vivo[J].Biol. Chem.2005,280:5892-5901.
[2]C.A. Ross, M.A. Poirier, Protein aggregation and neurodegenerative disease[J], Nat.Med.2004,10:10-17.
[3]R.M. Murphy, Peptide aggregation in neurodegenerative disease[J], Annu. Rev. Biomed. Eng.2002,4:155-174.
[4]V.N. Uversky, A.L. Fink, Conformational constraints for amyloid fibrillation:the importance of being unfolded[J], Biochim. Biophys. Acta.2004,1698:131-153.
[5]M.A.Chad, A.Mikhail, Karymov, K. A. Y. Lushnikov, M.V. Andrew,N. Uversky and L. L. Yuri. Protein Interactions and Misfolding Analyzed by AFM Force Spectroscopy[J].Mol. Biol.2005,354:1028-1042.
[6]C. M.Dobson, V.Bellotti, P.Mangione & M.Stoppini.Protein misfolding, evolution and disease[J]. Trends Biochem. Sci.1999,24:329-332.
[7]T. Pettersson, MD, and Y. T. Konttinen, MD.Amyloidosis—Recent Developments [J]. Semin Arthritis Rheum 2010,39:356-368.
[8]D.Jean, A.S Sipe.Review: History of the Amyloid Fibril[J]. Journal of Structural Biology,2000,130:88-98.
[9]P.Riccardo, G.Enrico and C.Amedeo.Pathways and Intermediates of Amyloid Fibril Formation[J].Mol. Biol.2007,374:917-924.
[10]C.M. Dobson, Principles of protein folding, misfolding and aggregation[J], Semin. Cell.Dev. Biol.2004,15:3-16.
[11]S.S.S.Wang, T.A. Good, An overview of Alzheimer's disease[J]. Chin. Ins. Chem. Eng.2005,36:533-559.
[12]C.A. Ross, M.A. Poirier, Protein aggregation and neurodegenerative disease[J], Nat.Med.2004,10:10-17.
[13]V.N. Uversky, A.L. Fink, Conformational constraints for amyloid fibrillation:the importance of being unfolded, Biochim. Biophys. Acta.2004,1698:131-153.
[14]E.L.Annette, V.Bente.Methods for structural characterization of prefibrillar inter-mediates and amyloid fibrils[J].FEBS Letters,2009,583:2600-2609.
[15]C. Louise, L.Serpell, S. Margaret, D. Merrill, G.A.Benson, M. B.Tennent, Pepys and E. F. Paul. The Protofilament Substructure of Amyloid Fibrils[J]. J. Mol. Biol.2000,300:1033-1039.
[16]D.L. Rymer, T.A. Good, The role of G protein activation in the toxicity of amyloidogenic Abeta-(1-40), Abeta-(25-35), and bovine calcitonin, J. Biol. Chem.2001,276:2523-2530.
[17]D.M. Walsh, A. Lomakin, G.B. Benedek, M.M. Condron, D.B. Teplow, Amyloid beta-protein fibrillogenesis. Detection of a protofibrillar intermediate[J]. Biol. Chem.1997,272:22364-22372.
[18]R.V.Ward, K.H. Jennings, R. Jepras,W. Neville, D.E. Owen, J. Hawkins, G. Christie, J.B. Davis, A. George, E.H. Karran, D.R. Howlett, Fractionation and characterization of oligomeric, protofibrillar and fibrillar forms of beta-amyloid peptide[J], Biochem.2000,348:137-144.
[19]J.I.Guijarro, M.Sunde, J.A Jones, I.D.Campbell and C.M.Dobson.Amloid fibril formation by an SH3 domain[J], Proc.Natl.Acal.Sci.U.S.A.1998.95:4224-4228.
[20]J.Brange, L.Andrsen, E.D.Laursen,G..Meyn and E.Rasmussen.Toward under-standing insulin fibrillation[J].Pharm.Sci.1997.86:571-525.
[21]C.E. Macphee,and C.M.Dobson.Chemical dissenction and reassembly of amloid fibril formed fragment of transthyretin[J].Mol.BIOL.2000,297:1203-1215.
[22]S.Ohnishi, A.Koide and S.Koide.Solution conformation and amyliod-like fibril formation of a polar peptide detived from a a-hairpin in the OspA single-layeer a-sheet[J].Mol.BIOL.2000.301:477-489.
[23]P.T. Lansbury Jr. Evolution of amyloid:what normal protein folding may tell us about fibrillogenesis and disease[J], Proc. Natl. Acad. Sci. USA 1999,96:3 342-3344.
[24]S.R. Ji, Y. Wu, S.F. Sui, Study of the correlation of secondary structure of betaamyloid peptide (Abeta40) with the hydrophobic exposure under different conditions[J], Gen. Physiol. Biophys.2002,21:415-427.
[25]C.Soto,Alzheimer's andpriondisease asdisorders of proteinconformation: implications for the design of novel therapeutic approaches [J]. Mol. Med.1999, 77:412-418.
[26]K.Hun Mok,Jenny Pettersson,Sten Orrenius,Catharina Svanborg. HAMLET, protein folding, and tumor cell death[J]. Biochemical and Biophysical Research Communications,2007,354:1-7.
[27]K.J.Pettersson, S. Aits, L.Gustafsson et al. Can misfolded proteins be beneficial? The HAMLET case [J]. Annals of Medicine,2009,41:162-176.
[28]N.U.Vladimir.Amyloidogenesis of Natively Unfolded Proteins [J].Curr Alzheimer Res.,2008,June 5(3):260-287.
[29]J.L.Helen and N.H.Philip.Systemic amyloidosis[J].Current Opinion in Pharmaco-logy,2006,6:214-220.
[30]A.L.Leheninger, D.L. Nelson, M.M.Cox. Principles of Biochemistry. [J]. New York:Worth Publishers.1933:180,312.
[31]Lysozyme.[EB/OL].http://lysozyme.co.uk/.
[32]E.Johan, P.Joubert. The use of lysozyme[EB/OL]. http://www.wynboer.co.za/ recentarticles/0201lyso.php3
[33]J. Y.Sun, U.Chibuike, E. A.Rotimi, J.P.Wu.Multifunctional peptides from egg white lysozyme[J].Food Research International 2010,43:848-855.
[34]R.Sarma,R.Bott.Crystallographic study of turkey egg white lysozyme and its complex with a disaccharide.J Mol Biol.1997,113:555.
[35]S.S.S.Wang, S.W. Chou, K.N.Liu,C.H.Wu.Effects of glutathione on amyloid fib-rillation of hen egg-white lysozyme[J]. International Journal of Biological Macromolecules,2009,45:321-329.
[36]M.C. Vaney, S.Maignan, M. Ries-Kautt, A.Ducriux, High-resolution structure (1.33 A)of a HEW lysozyme tetragonal crystal grown in the APCF apparatus. [J].D Biol. Crystallogr.1996,52:505-517.
[37]刘莹,孙荣丹,杨翔华,王丽.溶菌酶的应用现状.《饲料工业》·2006年第27卷第6期.
[38]F.Erica, P. P. Laureto, J. Zurdo, M. D.Christopher and F .Angelo. A Highly Amyloidogenic Region of Hen Lysozyme[J].Mol. Biol.2004,340:1153-1165
[39]Bfxqt.Glutathione[EB/OL]. http://www.doc88.com/p-99827123190.html 2010,11,19
[40]D. R. Francesco, et al. Ef ficacy an d safety of high-dose cisplatin an dcyclophosphamide with glut athione prot ection in the treatment of bulky advanced epithelial ovarian cancer[J]. Cancer Chemother Pharm acol,1990, 25:355-360.
[41]Y.Z.Fang, S.Yang, G.Wu, etal. Flee radicals, antioxidants,and nutrition[J].Nutrit ion,2002,18:872-879.
[42]M.G. Niroshini., I.G.Gregory and J.Claus.Multiple roles of cysteine in bio-catalysis[J].Biochemical and Biophysical Research Communications.2003, 300:1-4.
[43]张苏,王凤山.固定化原生质体转化合成L—半胱氨酸的研究.山东大学,学位论文.
[44]F.D.Gillin,L.S.Diamond.Attaclllnent of Entamoeba histolytica to glass in a defmed Maintenance medium:specific requirement for cysteine and ascorbic acid[J].Journal of Protozoology,1980,27(4):474-478
[45]M. R Nilsson.Techniques to study amyloid fibril formation in vitro[J]. Methods, 2004,34:151-160
[46]M.Biancalana,K.Makabe,A.Koide,S.Molecular mechanism of Thioflavin-T binding to the surface of β-richpeptide self-assemblies[J].Journal of Molecular Biology,2009.385(4):1052-1063.
[47]A.L. Fink.ANS.In T.E.Creighton,editor.The Encyclopedia of monomeric insulin [J].The Journal of Biology.1999.
[48]N.Berova,K.Nakanishi,R.W.Woody(Eds.).Circular Dichroism:Principles and Applications[M].R.New York:Wiley-VCH,20,877-879.
[49]D.H.A.Corre,C.H.I.Ramos.The use of circular dichroism spectroscopy to study protein folding, form and function[J]. African Journal of Biochemistry Research,2009,3(5):164-173.
[50]N.Sreerama& R.W.Woody.A self-consistent method for the analysis of protein secondary structure from circular dichroism[J].Anal.Biochem.1993,209:32-44.
[51]S.Hermann.Tricine-SDS-PAGE.Nature Protocols[J].2006.1(1):16-22.
[52]F.P.Marcelo and L.N.Claudia.Electrophoretic Analysis (Tricine-SDS-PAGE) [J].Acta Farm.Bonaerense.2002,21(1):57-60
[53]S.Raymond,L.Weintraub.Acrylamide gel as a supporting medium for zone electrophoresis. Science[J].1959,130:711.
[54]A.L.Shapiro,E.Vinuela, J.J.Maizel. Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels[J]. Biochem Biophys Res Commun.1967,28(5):815-820.
[55]H.L.Kristian, B.H.Mevik, E.O. Rukke, T.Almφy.Quantitative whole spectrum analysis with MALDI-TOF MS, Part Ⅰ:Measurement optimization[J]. Chemometrics and Intelligent Laboratory Systems.2009.96:210-218.
[56]F.Erica, P.L.Patrizia,Z.Jesus, M.D.Christopher and F.AAngelo.Highly Amyloid-ogenic Region of Hen Lysozyme[J]. J.Mol. Biol.2004,340:1153-1165
[57]L.N.Arnaudov, V.R.De. Thermally induced fibrillar aggregation of hen egg white lysozyme[J]. Biophysical Journal,2005,88:515-526.
[58]H.Akihito,A.Hiroyuki, and K.Akio.Amyloid Fibril Formation of Hen Lysozyme Depends on the Instability of the C-Helix (88-99)[J]. Biosci. Biotechnol. Biochem.,2008,72 (6):1523-1530.
[59]H. LeVine Ⅲ, Thioflavine T interaction with synthetic Alzheimer's disease beta-amyloid peptides:detection of amyloid aggregation in solution[J],.Protein. Sci. 1993,2:404-410.
[60]H.Daniel,A.Correaland H.Carlos,I.Ramos.The use of circular dichroism spectr-oscopy to study protein folding, form and function[J]. African Journal of Biochemistry Research,2009,3(5):164-173.
[61]M.R.Nilsson.Techniques to study amyloid fibril formation in vitro[J]. Methods, 2004,34:151-160.
[62]K.Young, O.H.Sam, R.G.Natalie,Y.Korlann, V. L.Elizabeth., R.C.Frank and W. Shimon.Efficient Site-Specific Labeling of Proteins via Cysteines[J]. Biocon-jugate Chem.2008,19:786-791.
[63]R.Vassilios,H.Rasmus, C.Lydia, R.E.Stephen.Separation and identification of hen egg protein isoforms using SDS-PAGE and 2D gel electrophoresis with MALDI-TOF mass spectrometry[J]. Food Chemistry 2006,99:702-710
[64]K.Sadamu, K.Tamie, K.Naoka, N.Satomi and M.Hiroyuki. Quantitative proteome analysis using D-labeled N-ethylmaleimide and 13C-labeled iodoacetanilide by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry[J]. Bioorganic & Medicinal Chemistry.2006,14:8197-8209.
[65]M. Monti, A. Amoresano, S. Giorgetti, V. Bellotti, P. Pucci. Limited proteolysis in the investigation of h2-microglobulin amyloidogenic and fibrillar states[J]. Biochimica et Biophysica Acta 2005,1753:44-50.
[66]R.Gramer,M.Saxton,K.Barnouin. Sample preparation of gel electrophoretically separated protein binding partners for analyais by volatile solubilizing agents[J]. Methods Mol Biol.24,261:499-510.
[67]王旭初,范鹏翔,李银心,等.一种适合与质谱分析的简化叫内酶解方法[J].植物生物与分子生物学学报,2007,33(5)：449-455.
[68]W.John & L.Sons,.Proteins structure and function[M].2005.
[69]A.L. Smoot, M. Panda, B.T. Brazil, A.M. Buckle, A.R. Fersht, P.M. Horowitz, The binding of bis-ANS to the isolated GroEL apical domain fragment induces the formation of a folding intermediate with increased hydrophobic surface not observed in tetradecameric GroEL[J], Biochemistry.2001,40:4484-4492.
[70]C.P. Liu, Z.Y. Li, G.C. Huang, S. Perrett, J.M. Zhou, Two distinct intermediates of trigger factor are populated during guanidine denaturation[J], Biochimie.2005, 87:1023-1031.
[71]I.Sirangelo,E.Bismuto,S.Tavassi,G..Irace.Apomyoglobin folding intermediates characterized by the hydrophobic fluorescent probe 8-anilino-1-naphthalene sulfonate[J], Biochim. Biophys. Acta.1998,1385:69-77.
[72]S.S.S.Wang,K.N.Liu,W.H.Lee. Effect of curcumin on the amyloid fibrillogenesis of hen egg-white lysozyme[J]. Biophysical Chemistry.2009,144:78-87.
[73]M. Dumoulin, J.R. Kumita, C.M. Dobson, Normal and aberrant biological self-assembly:insights from studies of human lysozyme and its amyloidogenic variants[J], Acc. Chem. Res.2006,39:603-610.
[74]M.B. Pepys, P.N. Hawkins, D.R. Booth, D.M. Vigushin, GA. Tennent, A.K. Soutar, N.Totty, O. Nguyen, C.C. Blake, C.J. Terry, et al., Human lysozyme gene mutations cause hereditary systemic amyloidosis[J], Nature.1993,362:553-557.
[75]I.A. Cotgreave, R.G. Gerdes, Glutathione-protein interactions:A molecular link between oxidative stress and cell proliferation? [J] Bio-chem. Biophys. Res. Commun.1998,242:1-9.
[76]F. Aslund, J. Beckwith.Bridge over troubled waters:Sensing stress by disulWde bond formation[J]. Cell.1999,96:751-753.
[77]M.S. Paget, M.J. Buttner, Thiol-based regulatory switches, Annu. Rev.Genet. 2003,37:91-121.
[78]K.R.Lynette, L.L.Barbara,V.S.Charles.Detection of reversible protein thiol modi Wcations in tissues[J]. Analytical Biochemistry.2006,358:171-184.
[79]B.Huang, J.He, J.Ren, X.Y.Yan, and C.M.Zeng. Cellular Membrane Disruption by Amyloid Fibrils Involved Intermolecular Disulfide Cross-Linking[J]. Biochemistry 2009,48:5794-5800.
[80]Y. Porat, A. Abramowitz, E. Gazit, Inhibition of amyloid fibril formation by polyphenols:structural similarity and aromatic interactions as a common inhibition mechanism[J], Chem. Biol. Drug. Des.2006,67:27-37.
[81]D.R. Howlett, A.E. Perry, F. Godfrey, J.E. Swatton, K.H. Jennings, C. Spitzfaden, H.Wadsworth, S.J.Wood, R.E.Markwell, Inhibition of fibril formation in beta-amyloid peptide by a novel series of benzofurans[J], Biochem.J.1999,340:283-289.
[82]W.E. Klunk, M.L. Debnath, A.M. Koros, J.W. Pettegrew, Chrysamine-G, a lipophilic analogue of Congo red, inhibits A beta-induced toxicity in PC 12 cells[J], Life Sci.1998,63:1807-1814.
[83]T. Tomiyama, A. Shoji, K. Kataoka, Y. Suwa, S. Asano, H. Kaneko, N. Endo, Inhibition of amyloid beta protein aggregation and neurotoxicity by rifampicin. Its possible function as a hydroxyl radical scavenger[J],Biol.Chem.1996,271 6839-6844.
[84]T. Tomiyama, H. Kaneko, K. Kataoka, S. Asano, N. Endo, Rifampicin inhibits the toxicity of pre-aggregated amyloid peptides by binding to peptide fibrils and preventing amyloid-cell interaction[J], Biochem. J.1997,322:859-865.
[85]A. Lomakin, D.S. Chung, G.B. Benedek, D.A. Kirschner, D.B. Teplow, On the nucleation and growth of amyloid beta-protein fibrils:detection of nuclei and quantitation of rate constants[J], Proc. Natl. Acad. Sci. U. S. A.1996,93: 1125-1129.
[86]S.J. Wood, L. MacKenzie, B. Maleeff, M.R. Hurle, R.Wetzel, Selective inhibition of Abeta fibril formation[J], J. Biol. Chem.1996,271:4086-4092.
[87]K. Ono, Y. Yoshiike, A. Takashima, K. Hasegawa, H. Naiki, M. Yamada, Potent anti-amyloidogenic and fibril-destabilizing effects of polyphenols in vitro: implications for the prevention and therapeutics of Alzheimer's disease[J]. Neurochem.2003,87:172-181.
[88]L.D. Estrada, C. Soto, Disrupting beta-amyloid aggregation for Alzheimer disease treatment, Curr. Top[J]. Med. Chem.2007,7:115-126.
[89]R. Liu,H.Barkhordarian,S.Emadi, C.B. Park, M.R. Sierks,Trehalose differentially inhibits aggregation and neurotoxicity of beta-amyloid[J].Neurobiol.2005,20 :74-81.
[90]F.G. De Felice,M.N. Vieira, L.M. Saraiva, J.D. Figueroa-Villar, J. Garcia-Abreu, R. Liu, L.Chang, W.L. Klein, S.T. Ferreira, Targeting the neurotoxic species in Alzheimer's disease:inhibitors of Abeta oligomerization[J], Faseb J.2004,18: 1366-1372.
[91]P. N.Dekhuijzen. Antioxidant properties of Nacetylcysteine:their relevance in relation to chronic obstructive pulmonary disease[J]. Eur. Respir. J.2004,23:629-636.
[92]Y.Ohhashi, Y.Hagihara, G.Kozhukh, M.Hoshino, K.Hasegawa, I.Yamaguchi, et al. The intrachain disulfide bond of β2-microglobulin is not essential for the immunoglobulin fold at neutral pH,but is essential for amyloid fibril formation at acidic pH[J]. Biochem.2002,131:45-52.
[93]D. P.Hong, M Gozu, K.Hasegawa, H.Naiki& Y. Goto.Conformation of β2-microglobulin amyloid fibrils analyzed by reduction of the disulfide bond[J]. Biol. Chem.2002,277:21554-21560.
[94]K. Ono, K. Hasegawa, H. Naiki, M. Yamada, Curcumin has potent anti-amyloidogenic effects for Alzheimer's beta-amyloid fibrils in vitro[J]. Neurosci. Res.2004,75:742-750.
[95]K. Ono, M. Yamada, Antioxidant compounds have potent anti-fibrillogenic and fibril-destabilizing effects for alpha-synuclein fibrils in vitro[J]. Neurochem. 2006,97:105-115.
[96]F. Yang, G.P. Lim, A.N. Begum, O.J. Ubeda,M.R. Simmons, S.S. Ambegaokar, P.P. Chen,R. Kayed, C.G. Glabe, S.A. Frautschy, G.M. Cole, Curcumin inhibits formation of amyloid beta oligomers and fibrils, binds plaques, and reduces amyloid in vivo[J].Biol. Chem.2005,280:5892-5901.