Journal of Textile Research ›› 2021, Vol. 42 ›› Issue (07): 46-53.doi: 10.13475/j.fzxb.20201006308

• Fiber Materials • Previous Articles     Next Articles

Separation and characterization of silk fibroin with different molecular weight

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

  1. 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 Online:2021-07-15 Published:2021-07-22
  • Contact: WANG Jiannan E-mail:wangjn@suda.edu.cn

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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

CLC Number: 

  • TS141.8

Tab.1

Formula of 10% resolving gel and 5% stacking gel"

试剂 体积/mL
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

Fig.1

SDS-PAGE electrophoretogram of silk fibroin seperated at different laminar flow rate. (a) Unseparated silk fibroin; (b) Laminar flow rate is 15 mL/h; (c) Laminar flow rate is 30 mL/h; (d) Laminar flow rate is 60 mL/h"

Fig.2

Shear rate-viscosity curves of silk fibroin"

Tab.2

Viscosity of silk fibroin at different shear ratesPa·s"

丝素蛋白编号 黏度
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

Tab.3

Mole percentage of each amino acid in silk fibroin%"

氨基酸种类 理论计算氨基酸摩尔分数 实验测得氨基酸摩尔分数
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

Fig.3

Electronegativity of silk fibroin"

Fig.4

Surface potential ratios of separated and unseparated silk fibroin"

Fig.5

Hydrophilic amino acid content in silk fibroin sample"

Fig.6

CD spectra of silk fibroin"

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