不同分子质量丝素蛋白的分离与表征

doi:10.13475/j.fzxb.20201006308

纺织学报 ›› 2021, Vol. 42 ›› Issue (07): 46-53.doi: 10.13475/j.fzxb.20201006308

不同分子质量丝素蛋白的分离与表征

丁梦瑶¹, 戴梦男¹, 李蒙¹, 刘苹¹, 徐晶晶¹, 王建南¹^,²()

1.苏州大学纺织与服装工程学院, 江苏苏州 215123
2.苏州大学现代丝绸国家工程实验室, 江苏苏州 215123

收稿日期:2020-10-27 修回日期:2021-04-13 出版日期:2021-07-15 发布日期:2021-07-22
通讯作者: 王建南
作者简介:丁梦瑶(1995—),女,硕士生。主要研究方向为功能纺织品。
基金资助:
国家自然科学基金项目(51873141);国家自然科学基金项目(51473108);江苏省自然科学基金项目(BK20181192);江苏省高等学校自然科学研究重大项目(18KJA540001)

Separation and characterization of silk fibroin with different molecular weight

DING Mengyao¹, DAI Mengnan¹, LI Meng¹, LIU Ping¹, XU Jingjing¹, WANG Jiannan¹^,²()

1. College of Textile and Clothing Engineering, Soochow University, Suzhou, Jiangsu 215123, China
2. National Engineering Laboratory for Modern Silk, Soochow University, Suzhou, Jiangsu 215123, China

Received:2020-10-27 Revised:2021-04-13 Published:2021-07-15 Online:2021-07-22
Contact: WANG Jiannan

摘要/Abstract

摘要：

为获得不同分子质量且分布相对集中的丝素蛋白,采用葡聚糖凝胶层析技术对再生丝素蛋白溶液进行梯度纯化、分离。通过使用合适的葡聚糖凝胶并控制层析流速,有效分离出不同分子质量的丝素蛋白,并进一步对获得的4组不同分子质量的丝素蛋白的黏度、电负性、氨基酸组成和分子构象进行分析。结果表明:丝素蛋白溶液的黏度和等电点随其分子质量的减小而减小,其电负性的提高主要源自于丝素重链被切断,且富含非重复区域的较小分子质量的肽链;4组不同分子质量的丝素蛋白均可自组装形成一定的β-折叠结构,分子质量越高的丝素蛋白越易形成稳定的分子构象。

关键词: 丝素蛋白, 凝胶层析, 分子质量, 两性性质, 分子构象

Abstract:

The sephadex gel chromatography was used for gradient purification and separation to obtain relatively centralized silk fibroin with different molecular weight distributions from regenerated silk fibroin. The silk fibroin with different molecular weights was separated effectively by using the appropriate sephadex gel and controlling the chromatographic flow rate.The viscosity, electronegativity, amino acid composition and molecular conformation of the preliminarily separated four silk fibroins were further analyzed. Results showed that the viscosity and isoelectric point of the silk fibroin solution decreased with the decrease of molecular weight. The increased electronegativity is mainly due to the silk fibroin heavy chain which were cut off into smaller peptide chains richly exists in non-repetitive regions. Each of the four silk fibroins in the study could form β-sheet structure by self-assemble, and the higher the molecular weight, the easier it was to form a stable molecular conformation.

Key words: silk fibroin, gel chromatography, molecular weight, amphoteric properties, molecular conformation

中图分类号:

TS141.8

丁梦瑶, 戴梦男, 李蒙, 刘苹, 徐晶晶, 王建南. 不同分子质量丝素蛋白的分离与表征[J]. 纺织学报, 2021, 42(07): 46-53.

DING Mengyao, DAI Mengnan, LI Meng, LIU Ping, XU Jingjing, WANG Jiannan. Separation and characterization of silk fibroin with different molecular weight[J]. Journal of Textile Research, 2021, 42(07): 46-53.

图/表 9

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参考文献 22

[1]	INOUE S, TANAKA K, ARISAKA F, et al. Silk fibroin of Bombyx mori is secreted, assembling a high molecular mass elementary unit consisting of H-chain, L-chain, and P25, with a 6:6:1 molar ratio[J]. The Journal of Biological Chemistry, 2000, 275(51):40517-40528. doi: 10.1074/jbc.M006897200
[2]	CHELAZZI D, BADILLO-SANCHEZ D, GIORGI R, et al. Self-regenerated silk fibroin with controlled crystallinity for the reinforcement of silk[J]. Journal of Colloid and Interface Science, 2020, 576:230-240. doi: 10.1016/j.jcis.2020.04.114
[3]	殷音, 郝云霞, 刘志武, 等. 不同条件制备的血管样蚕丝编织物小口径管状支架及其形态结构与细胞毒性测定[J]. 蚕业科学, 2015, 41(2):361-366.
	YIN Yin, HAO Yunxia, LIU Zhiwu, et al. Morphological structure and cytotoxicity of blood vessel-like small diameter silk fibroin-based tubular scaffold prepared under different conditions[J]. Science of Sericulture, 2015, 41(2):361-366.
[4]	QIAN K Y, SONG Y H, YAN X, et al. Injectable ferrimagnetic silk fibroin hydrogel for magnetic hyperthermia ablation of deep tumor[J]. Biomaterials, 2020, 259:120229.
[5]	WANG H Y, ZHANG Y Q, WEI Z G. Excess acetone extraction in silk protein solution greatly accelerates the regeneration progress of silk fibroin for desalting and purification[J]. International Journal of Biological Macromolecules, 2020, 146:588-595. doi: 10.1016/j.ijbiomac.2019.12.274
[6]	WANG Q, CHEN Q, YANG Y H, et al. Effect of various dissolution systems on the molecular weight of regenerated silk fibroin[J]. Biomacromolecules, 2013, 14(1):285-289. doi: 10.1021/bm301741q
[7]	CHO H J, KI C S, OH H, et al. Molecular weight distribution and solution properties of silk fibroins with different dissolution conditions[J]. International Journal of Biological Macromolecules, 2012, 51(3):336-341. doi: 10.1016/j.ijbiomac.2012.06.007
[8]	LEI C H, ZHU H L, LI J J, et al. Preparation and hemostatic property of low molecular weight silk fibroin[J]. Journal of Biomaterials Science (Polymer Edition), 2016, 27(5):403-418.
[9]	ZENG D M, PAN J J, WANG Q, et al. Controlling silk fibroin microspheres via molecular weight distribution[J]. Materials Science and Engineering: C, 2015, 50:226-233. doi: 10.1016/j.msec.2015.02.005
[10]	JIANG F J, LIU K, ZHAO M H, et al. Tunable high-molecular-weight silk fibroin polypeptide materials: fabrication and self-assembly mechanism[J]. ACS Applied Bio Materials, 2020, 3(5):3248-3259. doi: 10.1021/acsabm.0c00231
[11]	NULTSCH K, BAST L K, NÄF M, et al. Effects of silk degumming process on physicochemical, tensile, and optical properties of regenerated silk fibroin[J]. Macromolecular Materials and Engineering, 2018, 303(12):1800408. doi: 10.1002/mame.v303.12
[12]	WANG L P, LUO Z W, ZHANG Q, et al. Effect of degumming methods on the degradation behavior of silk fibroin biomaterials[J]. Fibers and Polymers, 2019, 20(1):45-50. doi: 10.1007/s12221-019-8658-9
[13]	KIM H J, KIM M K, LEE K H, et al. Effect of degumming methods on structural characteristics and properties of regenerated silk[J]. International Journal of Biological macromolecules, 2017, 140:294-302. doi: 10.1016/j.ijbiomac.2019.08.035
[14]	吴疆, 童应凯, 杨红澎. 生物分离实验技术[M]. 北京: 化学工业出版社, 2009: 229-230.
	WU Jiang, TONG Yingkai, YANG Hongpeng. Bioseparation technology[M]. Beijing: Chemical Industry Press, 2009: 229-230.
[15]	李锦杰, 赵东旭, 马增红. 中分子量可溶性丝素蛋白的制备的研究[EB/OL]. 北京:中国科技论文在线, 2008 [2020-10-23]. http://www.paper.edu.cn/releasepaper/content/200806-80.
	LI Jinjie, ZHAO Dongxu, MA Zenghong. Preparation of water-soluber silk fibroin with moderate molecuar[EB/OL]. Beijing: Sciencepaper Online, 2008 [2020-10-23]. http://www.paper.edu.cn/releasepaper/content/200806-80.
[16]	WANG J N, YAN S Q, LU C D, et al. Biosynjournal and characterization of typical fibroin crystalline polypeptides of silkworm Bombyx mori[J]. Materials Science and Engineering: C, 2009(4):1321-1325.
[17]	WANG Z Q, YANG H W, LI W, et al. Effect of silk degumming on the structure and properties of silk fibroin[J]. Journal of The Textile Institute, 2019, 110(1):134-140. doi: 10.1080/00405000.2018.1473074
[18]	ZHOU C Z, CONFALONIERI F, JACQUET M, et al. Silk fibroin: structural implications of a remarkable amino acid sequence[J]. Proteins-structure Function & Bioinformatics, 2010, 44(2):119-122.
[19]	YAMAGUCHI K, KIKUCHI K, TAKAGI T, et al. Primary structure of the silk fibroin light chain determined by cDNA sequencing and peptide analysis[J]. Journal of Molecular Biology, 1989, 210(1):127-139. doi: 10.1016/0022-2836(89)90295-7
[20]	MARTINE C, PIERRE C, JEAN-CLAUDE P. Complete nucleotide sequence of the gene encoding the Bombyx mori silk protein P25 and predicted amino acid sequence of the protein[J]. Nuclc Acids Research, 1986, 14(15):6341-6342.
[21]	MORRISETT J D, DAVID J S, POWNALL H J, et al. Interaction of an apolipoprotein (apoLP-alanine) with phosphatidylcholine[J]. Biochemistry, 1973, 12(7):1290-1299. doi: 10.1021/bi00731a008
[22]	KANG Z, WANG Y N, XU J J, et al. An RGD-containing peptide derived from wild silkworm silk fibroin promotes cell adhesion and spreading[J]. Polymers, 2018(10):1193-1207.

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试剂	体积/mL
试剂	灭菌水	30%丙烯酰胺溶液	1.5 mol/L Tris-HCl (pH值为8.8)缓冲液	1 mol/L Tris-HCl (pH值为6.8)缓冲液	10%SDS 溶液	10%过硫酸铵溶液	TEMED
10%分离胶	4.0	3.3	2.5	—	0.10	0.10	0.004
5%浓缩胶	2.1	0.5	—	0.38	0.03	0.03	0.003

丝素蛋白编号	黏度
丝素蛋白编号	最大值	起始值	剪切速率为4 s^-1处
SF0	11.60	6.77	1.31
SF1	16.23	7.60	1.55
SF2	9.62	6.92	1.33
SF3	8.64	6.72	1.17
SF4	4.87	1.84	0.56

氨基酸种类	理论计算氨基酸摩尔分数
氨基酸种类	H链	L链	P25链	SF分子	SF0	SF1	SF2	SF3	SF4
天冬氨酸Asp	0.48	6.49	6.36	0.80	1.59	1.56	1.55	1.49	1.59
苏氨酸Thr	0.89	3.05	5.45	1.02	0.96	0.94	0.92	1.19	0.97
丝氨酸Ser	12.07	9.54	6.36	11.91	10.77	10.68	10.61	11.04	10.84
谷氨酸Glu	0.57	1.91	3.18	0.65	1.53	1.51	1.55	2.39	1.76
甘氨酸Gly	45.89	8.40	4.09	43.84	44.23	44.09	43.69	43.58	43.70
丙氨酸Ala	30.27	13.74	7.27	29.34	30.53	30.69	31.02	30.75	31.37
半胱氨酸Cys	0.10	1.15	4.09	0.17	0.19	0.26	0.34	0.00	0.26
缬氨酸Val	1.84	7.25	5.45	2.12	1.98	2.08	2.12	3.28	2.11
蛋氨酸Met	0.08	0.76	0.91	0.11	0.00	0.05	0.06	0.00	0.00
异亮氨酸Ile	0.25	8.02	6.36	0.65	0.45	0.47	0.46	0.30	0.44
亮氨酸Leu	0.13	7.63	10.00	0.55	0.38	0.31	0.34	0.00	0.44
酪氨酸Tyr	5.26	4.20	4.55	5.21	4.97	4.95	4.93	4.48	4.93
苯丙氨酸Phe	0.55	3.05	6.82	0.71	0.70	0.68	0.69	1.19	0.70
赖氨酸Lys	0.23	1.91	3.18	0.33	0.25	0.26	0.23	0.30	0.26
组氨酸His	0.10	1.91	3.64	0.20	0.19	0.21	0.23	0.00	0.26
精氨酸Arg	0.27	4.20	5.91	0.49	0.32	0.26	0.29	0.00	0.35
脯氨酸Pro	0.27	3.44	5.45	0.45	0.96	0.99	0.97	0.00	0.00
色氨酸Trp	0.21	0.76	1.36	0.24	0.00	0.00	0.00	0.00	0.00
天冬酰胺Asn	0.38	6.87	5.91	0.72	0.00	0.00	0.00	0.00	0.00
谷氨酰胺Gln	0.19	5.73	3.64	0.47	0.00	0.00	0.00	0.00	0.00
合计	100.00	100.00	100.00	100.00	100.00	100.00	100.00	100.00	100.00

不同分子质量丝素蛋白的分离与表征

Separation and characterization of silk fibroin with different molecular weight

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