Thin-layer chromatography of the sericin,fatty waxes,and fibroin of natural silk

The method of thin-layer and high-performance thin-layer chromatography is proposed for the investigation of the isolation of sericin, fatty waxes, and fibroin from a natural silk cocoon. The chromatographic fractionation of fibroin according to molecular masses has been studied.Institute of the Chemistry and Physics of Polymers, Academy of Sciences of the Republic of Uzbekistan, Tashkent. fax (3712) 44 26 61. Translated from Kbimiya Prirodnykh Soedinii, No. 4,pp. 588–590, July–August, 1995. Original article submitted October 17, 1994.     

Structure and molecular conformation of tussah silk fibroin films: Effect of methanol

Structural changes of tussah (Antheraea pernyi) silk fibroin films treated with different water-methanol solutions at 20°C were studied as a function of methanol concentration and immersion time. X-ray diffraction measurements showed that the α-helix structure, typical of untreated tussah films, did not change for short immersion times (2 min), regardless of methanol concentration. However, crystallization to β-sheet structure was observed following immersion of tussah films for 30 min in methanol solutions ranging from 20 to 60% (v/v). IR spectra of tussah films untreated and methanol treated for 2 min exhibited strong absorption bands at 1265, 892, and 622 cm^?1, typical of the α-helix conformation. The intensity of the bands assigned to the β-sheet conformation (1245, 965, and 698 cm^?1) increased for the sample treated with 40% methanol for 30 min. Raman spectra of tussah films with α-helix molecular conformation exhibited strong bands at 1657 (amide I), 1263 (amide III), 1106, 908, 530, and 376 cm^?1. Following α → β conformational transition, amide I and III bands shifted to 1668, and to 1241, 1230 cm^?1, respectively. The band at 1106 cm^?1 disappeared and new bands appeared at 1095 and 1073 cm^?1, whereas the intensity of the bands at 530 and 376 cm^?1 decreased significantly. ©1995 John Wiley & Sons, Inc.     

Mechanisms of silk fibroin sol-gel transitions

Silk fibroin sol-gel transitions were studied by monitoring the process under various physicochemical conditions with optical spectroscopy at 550 nm. The secondary structural change of the fibroin from a disordered state in solution to a beta-sheet-rich conformation in the gel state was assessed by FTIR and CD over a range of fibroin concentrations, temperatures, and pH values. The structural changes were correlated to the degree of gelation based on changes in optical density at 550 nm. No detectable changes in the protein secondary structure (FTIR, CD) were found up to about 15% gelation (at 550 nm), indicating that these early stages of gelation are not accompanied by the formation of beta-sheets. Above 15%, the fraction of beta-sheet linearly increased with the degree of gelation. A pH dependency of gelation time was found with correlation to the predominant acidic side chains in the silk. Electrostatic interactions were related to the rate of gelation above neutral pH. The overall independencies of processing parameters including concentration, temperature, and pH on gel formation and protein structure can be related to primary sequence-specific features in the molecular organization of the fibroin protein. These findings clarify aspects of the self-assembly of this unique family of proteins as a route to gain control of material properties, as well as for new insight into the design of synthetic silk-biomimetic polymers with predictable solution and assembly properties.     

Interfacial rheology of natural silk fibroin at air/water and oil/water interfaces

The interfacial viscoelastic behavior of natural silk fibroin at both the air/water and oil/water interfaces is reported. This natural multiblock copolymer is found to be strongly amphiphilic and forms stable films at these interfaces. The result is an interfacial layer that is rheologically complex with strong surface elastic moduli that are only slightly frequency-dependent. The kinetics of surface viscoelastic evolution are reported as functions of time for various concentrations of the spread films. Films deposited by Langmuir-Blodgett deposition were studied by scanning electron microscopy (SEM) to reveal a fibrous structure at the interface. The production of stable O/W emulsions by silk fibroin further confirms the generation of the elastic films at the oil/water interfaces.     

Structure and molecular conformation of tussah silk fibroin films: Effect of heat treatment

Structural changes of tussah (Antheraea pernyi) silk fibroin films induced by heat treatment were studied as a function of the treatment temperature in the range 200–250°C. The DSC curve of tussah films with α-helix molecular conformation displayed characteristic endo and exo peaks at 216 and 226°C, respectively. These peaks first weakened and then completely disappeared after heating at 230°C. Accordingly, the TMA thermal shrinkage at 206°C disappeared when the films were heated at 230°C. The onset of weight loss was monitored at 210°C by means of TG measurements. X-ray diffraction profiles gradually changed from α-helix to β-sheet crystalline structure as the treatment temperature increased from 200 to 250°C. On raising the heating temperature above 200°C, the intensity of IR and Raman bands characteristic of β-sheet conformation increased in the whole ranges of amide and skeletal modes. The sample treated at 200°C showed a spectral pattern intermediate between α-helix and β-sheet molecular conformation. The IR marker band for random coil structure, still detectable at 200°C, disappeared at higher treatment temperatures. Spectral changes attributable to the onset of thermal degradation appeared at 230°C. © 1997 John Wiley & Sons, Inc. J Polym Sci B: Polym Phys 35: 841–847, 1997     

Simple preparation and characteristics of silk fibroin microsphere

We investigated the biomaterial and pharmaceutical utility of pure silk fibroin (SF) protein as a possible for separation, using Sephadex G-25 gel filtration chromatography and simply preparing SF microsphere particles (SFMP) by spray dryer. Also, some of its physicochemical properties and morphology were investigated. Obtaining microspheres and/or submicronic particles by spray dryer method was accelerated or completed with the transition from the random coil to the β-sheet structure during spray dryer treatment. It was identified by the basic Fourier transform infrared spectroscopy of SFMP. The various pH range of SFMP’s swelling ratio is dependent on the pH of the solution, not on the occurred gelation. Morphologically, SFMP was spherical in shape, and particles, average 2±10 μm in size, were observed by scanning electron microscope and particle analyzer, respectively. The average molecular weight (M_W) of pure SF protein dissolved in calcium chloride is about 61,500 g/mol as measured by gel permeation chromatography.     

Gelation behavior of Antheraea pernyi silk fibroin

The sol-gel transition behavior of Antherae pernyi silk fibroin(Ap-SF) has not been systematically investigated.In this work,the influence of environmental temperature,pH,the concentration of Ap-SF,K+ and Ca2+ on the gelation time,and the structural changes of Ap-SF in sol-gel transformation were studied.The results indicated that the gelation time of the Ap-SF aqueous solution decreased with the increase of the Ap-SF concentration and environmental temperature.The sol-gel transformation of Ap-SF was much m...     

Salt-catalyzed reaction between styrene oxide and silk fibroin

The addition reaction of styrene oxide (StO) with silk fibroin was studied in the presence of various salts in different solvents at 45–75°C. Some water was required to make StO react with silk padded with various salt solutions. The reaction rate increased with the salt concentration and reached a maximum value at a certain concentration of the salt. Padding with solutions of thiosulfate, cyanide, thiocyanate, bicarbonate, or carbonate resulted in high add-ons (to 65 mole/10⁵ g) and low solubilities in HCl and NaOH aqueous solutions. The weight gains increased with the epoxide concentration and reached a constant value at a certain concentration of StO solution in ethanol, while they decreased slightly with epoxide concentration over 10% of StO solution in n-hexane. Histidine, lysine, arginine, tyrosine, and aspartic and glutamic acids were found to react. The reaction rate decreased with increasing solubility parameter of the solvent used, reached a minimum value about at 10 or at the solubility parameter of the epoxide, and then increased with the parameter. The StO–silk reaction may depend on the distribution of StO between aqueous salt and an organic solvent phases, and on the swelling of silk fiber in different aqueous salt solutions or in various organic solvents. The mechanism for this epoxide-silk reaction and the reactivity difference between StO and phenyl glycidyl ether toward silk fibroin are discussed in the light of the observed phenomena.     

Structure and properties of composites compression‐molded from silk fibroin powder and waterborne polyurethane

The degummed silk filament was pulverized with a home‐made machine to obtain the silk fibroin (SF) powder with the diameter of around 3 µm. The resulting SF powder was blended with waterborne polyurethane (WPU) aqueous dispersion, and then was dried and compression‐molded to prepare novel blended materials with improved miscibility and mechanical properties. WPU acted as a plasticizer and one of the components for the blends during the compression‐molded process. The structure, morphology, and properties of the blended films were investigated. The results indicated that β‐sheet of SF existed in the blended films. The SEM images showed that the cross‐section of the blended films exhibited an overall homogeneous morphology. Furthermore, the transmission electron microscope observation exhibited that some sphere‐like SF particles were well dispersed in the WPU matrix. The hydrogen bond interaction between SF and WPU in the blended films led to an increase of the glass transition temperature for the soft segment of WPU in the blended films. The blended films showed an improved Young's modulus and tensile strength from 1.2 to 288.9 MPa and 0.3 to 16.5 MPa, respectively, with the increasing of SF up to a content of 70 wt%. The hydrogen‐bonding interactions existing in SF and WPU and compression molding method played the important role in improving the miscibility and mechanical properties of the blended films. Copyright © 2011 John Wiley & Sons, Ltd.     

Physical and chemical properties of tussah silk fibroin films

Physical and chemical structure, as well as thermal behavior of solution-cast regenerated films, prepared from tussah (Antheraea pernyi) silk fibroin, were compared with those of solution-cast native films, in order to ascertain whether treatment (degumming, dissolution) used for preparation affected their properties. Regenerated fibroin films exhibited a higher thermal stability than native ones, as shown by differential scanning calorimetry, thermomechanical analysis, and dynamic mechanical behavior. Glass transition temperature and other relevant thermal transitions of the regenerated silk specimen shifted to higher temperatures compared with those of native specimen. Molecular conformation and crystalline structure did not show significant differences between the two kinds of silk films. Amino acid composition and molecular weight, however, distribution changed markedly after dissolving tussah silk fibroin fiber in concentrated LiSCN in polypeptide size was the main features for the regenerated silk fibroin. © 1994 John Wiley & Sons, Inc.     

The influence of UV radiation on silk fibroin

An investigation into the influence of UV-irradiation on regenerated silk fibroin dissolved in water was carried out using UV-Vis and fluorescence spectroscopy. It was found that the absorption of regenerated silk fibroin in solution increased during UV-irradiation of the sample, most notably between 250 and 400 nm. Moreover, after UV-irradiation a wide peak emerged between 290 and 340 nm with maximum at about 305 nm. The new peak suggests that new photoproducts are formed during UV-irradiation of regenerated silk fibroin.The fluorescence of regenerated silk fibroin was observed at 305 nm, at 480 nm and at 601 nm after excitation at 275 nm. UV-irradiation caused fluorescence fading at 305 nm and at 601 nm. The increase of fluorescence was observed at 480 nm, probably due to formation of new photoproducts. After excitation at 305 nm the fluorescence of regenerated silk fibroin was observed at 340 nm and at 400 nm. UV-irradiation caused fluorescence fading at 340 nm. FTIR spectroscopy showed that primary structure of regenerated silk fibroin was not significantly affected by UV radiation. SDS-PAGE chromatography showed alterations of molecular weight of silk after UV exposure.     

Biomaterial coatings by stepwise deposition of silk fibroin

A completely aqueous, stepwise deposition process with Bombyx mori silk fibroin for the assembly of nanoscale thin film coatings is reported the first time. The focus of this work was to develop an understanding of the control of this deposition process and to characterize the films formed from a physicochemical perspective. The deposition process was monitored by UV spectrophotometry and research quartz crystal microbalance. Both absorbance and film thickness correlated linearly with the number of silk fibroin layers deposited, analogous to multilayered materials fabricated from conventional polyelectrolytes. The polymer adsorption process was stable and reproducible, with control of a single layer thickness ranging from a few to tens of nanometers, determined by the concentrations of silk fibroin, salt concentration in the dipping solution, and method of rinsing. The driving force for the assembly of silk fibroin onto the substrate was primarily hydrophobic interactions, while some electrostatic interactions were also involved. The difference with this approach from traditional polyelectrolyte layer-by-layer techniques is that an intervening drying step is used to control the structure and stability of the self-assembled silk fibroin. The assembled films were stable under physiological conditions and supported human bone marrow stem cell adhesion, growth, and differentiation. This approach offers new options to engineer biomaterial coatings as well as bulk materials with control of both interfacial properties conducive to specific cellular or tissue responses and the potential to entrap and deliver labile molecules or other components due to the all-aqueous process described.     

Preparation and characterization of conjugates of silk fibroin and chitooligosaccharides

With the aim of the functionalization of silk fibroin (SF), conjugates of SF and polycationic chitooligosaccharides (COS) were prepared by the chemical modification of SF with cyanuric chloride (CY)-activated COS (COS-CY). The ¹H NMR spectrum of the reaction product between a model compound D-glucosamine and CY suggested that the COS-CY modifier was synthesized by the reaction of the amino group and the terminal anomeric hydroxyl group in COS, with the chlorine atom of CY. The ¹H NMR spectrum and amino acid analysis of the conjugates (COS-CY-SF) clarified that the tyrosine and lysine residues of SF reacted with a second chlorine atom of the triazine ring of the modifier. On the basis of the results of the hexosamine determination and the amino acid analysis of COS-CY-SF, it is estimated that COS-CY-SF consists of 38 wt% COS, 8 wt% CY, and 54 wt% SF. The absorbance at 600 nm as a function of pH for COS-CY-SF and SF indicated that the introduction of a large amount of hexosamine made SF amphiphilic and more water-soluble at lower pH values. The COS-CY-SF conjugates retarded the growth of Escherichia coli after incubation for 24 h at a conjugate concentration of 0.6% (w/v), while SF did not retard the growth at a SF concentration of 0.7% (w/v).     

Determination of the critical conditions for fiber formation by the fibroin of natural silk by polarization-optical methods

The processes of fiber formation by the fibroin of natural silk have been studied by the methods of birefringence in a longitudinal hydrodynamic field and optical rotatory dispersion. The experiments were performed with the direct drawing of a fiber from the secretion of the silk glands of the silkwormBombyx mori. The position of appearance of a longitudinal hydrodynamic field within the gland has been detected and experimental results have been obtained which permit an evaluation of the critical condition for the - structural transition of the fibroin chains.Institute of the Chemistry and Physics of Polymers, Academy of Sciences of the Republic of Uzbekistan, Tashkent, fax (3712) 44 26 61. Translated from Khimiya Prirodnykh Soedinenii, No. 4, pp. 623–627, July–August, 1997.     

Structure and structural changes of the silk fibroin from Samia cynthia ricini using nuclear magnetic resonance spectroscopy

The structure of silk fibroin from a wild silkworm, S. c. ricini, the amino acid sequence of which consists of repeated poly-Ala and Gly-rich regions, was examined by using solution and solid-state NMR methods. The structural transition of the silk fibroin in aqueous solution was monitored by using 13C solution NMR spectroscopy as a function of temperature. The fast exchange with respect to the chemical shift between the helix and coil conformations was observed in the poly-Ala region and the slow conformational change from alpha-helix to random coil was observed for the Gly residue adjacent to the N-terminal Ala residue of the poly-Ala region. The torsion angles of several Ala and Gly residues in the model peptide, GGAGGGYGGDGG(A)12GGA-GDGYGAG, were determined by the conformation-dependent 13C chemical shifts, rotational echo double resonance (REDOR) and 2D spin-diffusion NMR methods. The solid-state NMR analysis leads to the precise silk structure before spinning, where the poly-Ala sequence takes a typical alpha-helix pattern with a tightly winded helical structure at both terminal regions of the poly-Ala sequence. This is expected to stabilize the alpha-helical structure of the poly-Ala region in S. c. ricini silk fibroin from the silkworm.