蚕丝蛋白溶液的聚集和构象变化研究
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摘要
蚕丝蛋白作为一种天然动物纤维,具有优良的综合力学性能,良好的生物相容性和较低的生产成本,在纺织、国防、医药、食品和美容等诸多领域中具有较高的应用价值和良好的前景。蚕丝的成丝机理和构象转变成为目前研究的重点。
本文首先研究了不同途径提取的丝素蛋白的完整性。采用凝胶电泳定性的表征了再生丝素蛋白和丝腺丝素蛋白的水解情况。结果表明:第一,再生丝素蛋白在提取过程中会发生部分水解,其水解程度随溶解温度的提高和溶解时间的延长而加剧,并随溶剂体系的不同而变化。第二,中部丝腺的丝素蛋白没有发生水解现象,只存在完整的重链和轻链,是研究蛋白质构象转变的良好基质,而从后部丝腺中提取的蛋白含有较多的杂质。
本文通过圆二色谱、荧光光谱研究了再生丝素蛋白的溶液构象转变。圆二色谱的结果表明,再生丝素蛋白发生了由无规线团向β-折叠的构象转变,其动力学经历了两个不同速率的过程。色氨酸(Trp)残基的荧光发射谱发生蓝移,表明该残基在构象转变过程中,有亲水的蛋白质表面转移到疏水的蛋白质内部,其峰强有先减弱后增强的过程。
本文采用圆二色谱、荧光光谱和动态光散射研究了丝腺丝素溶液的构象转变和聚集情况。圆二色光谱表明,丝腺丝素蛋白溶液也会发生从无规线团向β-折叠的构象转变,随蛋白质浓度的增加,其构象转变速率加快。丝腺丝素蛋白的整个构象转变过程可以分为三个阶段,诱导(induction)、增长(growth)和稳定(stabilization)。与再生丝素的荧光光谱类似,丝腺丝素蛋白的Trp残基峰也有明显的蓝移现象。不同的是,丝腺丝素蛋白的荧光峰强不存在一个先减弱后增强的过程,较为均一。丝腺丝素蛋白的动态光散射结果表明,丝素蛋白的构象转变过程伴随着蛋白质分子的聚集,其最终的聚集体尺寸为2微米左右。
Because of its excellent comprehensive mechanical properties and bio-compatibilities, Silk, as on kind of nature animal fiber, has widely applications and potentials in the fields of textile, defense, medicine, food and cosmetic industry. Its formation mechanism and conformation change attract many attentions in the scientific area.
The current thesis investigates the integrity of silk fibroin extracted from solid silk fiber and silkworm gland by the measurement of protein molecular weight using SDS-PAGE. The results indicate that the regeneration process of silk fibroin for solid silk fiber will result in partial degradation, which depends on the temperature, time, degumming and dissolving agent. Furthermore, the silk fibroin extracted from the middle division of silkworm gland is intact and No degradation can be observed. The posterior division content, however, has many impurities.
Circular Dichroism Spectrometer (CD) and Fluorescence Spectrometer (FS) have been used to monitor the conformation change of regenerated silk fibroin (RSF) in aqueous solution. CD results suggest that RSF undergoes a conformation transition from random coil to P-sheet. The transition kinetics consists two different rate sections. The blue shift of Tryptophan (Trp) emission peek indicates that a micro-environment change occur during the conformation transition.
Gland Silk Fibroin (GSF) solution conformation transition and aggregation have been investigated by the combination of CD, FS and Dynamic Light Scattering (DLS). Similar as the CD and FS results of RSF, the conformation change from random coil toβ-sheet and micro-environment change of Trp residue can be observed from GSF solution by CD and FS. With increasing the protein concentration, the transition speed increase. The whole transition kinetics can be divided to three individual sections: induction, growth and stabilization. Accompanied with protein conformation change, the aggregation of protein molecules has been proved by DLS to a final size of 2μm.
本文首先研究了不同途径提取的丝素蛋白的完整性。采用凝胶电泳定性的表征了再生丝素蛋白和丝腺丝素蛋白的水解情况。结果表明:第一,再生丝素蛋白在提取过程中会发生部分水解,其水解程度随溶解温度的提高和溶解时间的延长而加剧,并随溶剂体系的不同而变化。第二,中部丝腺的丝素蛋白没有发生水解现象,只存在完整的重链和轻链,是研究蛋白质构象转变的良好基质,而从后部丝腺中提取的蛋白含有较多的杂质。
本文通过圆二色谱、荧光光谱研究了再生丝素蛋白的溶液构象转变。圆二色谱的结果表明,再生丝素蛋白发生了由无规线团向β-折叠的构象转变,其动力学经历了两个不同速率的过程。色氨酸(Trp)残基的荧光发射谱发生蓝移,表明该残基在构象转变过程中,有亲水的蛋白质表面转移到疏水的蛋白质内部,其峰强有先减弱后增强的过程。
本文采用圆二色谱、荧光光谱和动态光散射研究了丝腺丝素溶液的构象转变和聚集情况。圆二色光谱表明,丝腺丝素蛋白溶液也会发生从无规线团向β-折叠的构象转变,随蛋白质浓度的增加,其构象转变速率加快。丝腺丝素蛋白的整个构象转变过程可以分为三个阶段,诱导(induction)、增长(growth)和稳定(stabilization)。与再生丝素的荧光光谱类似,丝腺丝素蛋白的Trp残基峰也有明显的蓝移现象。不同的是,丝腺丝素蛋白的荧光峰强不存在一个先减弱后增强的过程,较为均一。丝腺丝素蛋白的动态光散射结果表明,丝素蛋白的构象转变过程伴随着蛋白质分子的聚集,其最终的聚集体尺寸为2微米左右。
Because of its excellent comprehensive mechanical properties and bio-compatibilities, Silk, as on kind of nature animal fiber, has widely applications and potentials in the fields of textile, defense, medicine, food and cosmetic industry. Its formation mechanism and conformation change attract many attentions in the scientific area.
The current thesis investigates the integrity of silk fibroin extracted from solid silk fiber and silkworm gland by the measurement of protein molecular weight using SDS-PAGE. The results indicate that the regeneration process of silk fibroin for solid silk fiber will result in partial degradation, which depends on the temperature, time, degumming and dissolving agent. Furthermore, the silk fibroin extracted from the middle division of silkworm gland is intact and No degradation can be observed. The posterior division content, however, has many impurities.
Circular Dichroism Spectrometer (CD) and Fluorescence Spectrometer (FS) have been used to monitor the conformation change of regenerated silk fibroin (RSF) in aqueous solution. CD results suggest that RSF undergoes a conformation transition from random coil to P-sheet. The transition kinetics consists two different rate sections. The blue shift of Tryptophan (Trp) emission peek indicates that a micro-environment change occur during the conformation transition.
Gland Silk Fibroin (GSF) solution conformation transition and aggregation have been investigated by the combination of CD, FS and Dynamic Light Scattering (DLS). Similar as the CD and FS results of RSF, the conformation change from random coil toβ-sheet and micro-environment change of Trp residue can be observed from GSF solution by CD and FS. With increasing the protein concentration, the transition speed increase. The whole transition kinetics can be divided to three individual sections: induction, growth and stabilization. Accompanied with protein conformation change, the aggregation of protein molecules has been proved by DLS to a final size of 2μm.
引文
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[2]胡玉洁主编.天然高分子材料改性与应用.化学工业出版社.北京.2003.
[3]平林洁,陈开利.绢の食品化.蚕丝科学と技术(日),30(1):43-45,1991
[4]Gregory H,et cl.Biomaterials 24:401-416,2004.
[5]浙江农业大学主编.《蚕体解剖生理学》,农业出版社,北京,1980.
[6]David Kaplan.Silk Polymers:Materials Science and Biotechnology,ACS,292-310.
[7]Shimura,K.Akai,H.,Iizuka,E.,Garel,J.P.,and Ikekawa,N.Experientia,39(5):441-550,1983.
[8]Magoshi,J.,Magoshi Y.,and Nakamura,S.,Crystallization,Liquid Crystal,and Fiber Formation of Silk Fibroin,J.Polym.Sci.:Appl.Polym.Symp.,41:187-204,1985.
[9]Kerkam,K.,Viney,C.,Kaplan,D.,and Lombardi,S.,Liquid Crystallinity of Natural Silk Secretions,Nature,349:596-598,1991.
[10]Viney,C.,Huber,A.E.,Dunaway,D.L.,Case,S.T.,and Kaplan,D.L.,L.Processing Natural and Reconstituted Silk Solutions under Equilibrium and Non-Equilibrium Conditions,Mat.Res.Soc.Symp.Proc.,292:211-217,1992.
[11]Viney,C.,Huber,A.E.,Dunaway.D.L.,Kerkam,K.,and Case,S.T.Optical Characterization of Silk Secretions and Fibers.in:Silk Polymers.Materials Science and Biotechnology,ACS Symposium Series 544;American Chemical Society:Washington D.C.,120-136,1994.
[12] Knight, D. and Vollrath, F., Hexagonal Columnar Liquid Crystal in the Cells Secreting Spider Silk, Tissue & Cell, 31 (6): 617-620,1999.
[13] Lotz, B. and Cesari, F.C., The Chemical and the Crystalline Structures of Bombyx mori Silk Fibroin, Biochimie, 61: 205-214,1979.
[14] Lucas, F., Shaw, J.R.B., and Smith, S.G., Extracellualr Fibrous Proteins: The Silks, Biochem. J., 66: 468-482,1957.
[15] Lucas, F. and Rudall, K.M. Extracellualr Fibrous Proteins: The Silks. In: Comprehensive Biochemistry, Ed. Florkin, M. and Stota, E.H. 26B, Elsevier, Amsterdam, 475-558,1968.
[16] Kay, L.M. and Schroeder, W.A., The Chromatographic Separation and Identification of Some Peptides in Partial Hydrolysates of Silk Fibroin, J. Am. Chem. Soc, 76: 3564-3568,1954.
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