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 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.     

Thermal properties of bombyx mori and several wild silkworm silks Phase transition of liquid silk

The thermal properties of liquid silk from domestic and wild silkworms are investigated. Liquid silks obtained from the silk gland of the domesticated silkworm, Bombyx mori and four wild silkworms, Samia cynthia ricini, Dictyoploca japonica, Antheraea pernyi and Antheraea yamamai were used. The DSC curves for the liquid silk from the domestic silkworm have weak endothermic peaks corresponding to the breaking of hydrogen bonds in the β-form or to the untangling of physical network. The DSC curves for the wild silkworm silks, however, show clear exothermic peaks corresponding to a phase transition from the α-helix conformation to the β-form. Liquid silk from all the different silkworms undergoes a characteristic irreversible phase transition. This revised version was published online in July 2006 with corrections to the Cover Date.     

1H pulsed NMR study of bombyx mori silk fibroin: Dynamics of fibroin and of absorbed water

The dynamical behavior of the Bombyx mori silk fibroin chain and of absorbed water in silk fiber, film, and powder has been studied by ¹H pulsed nuclear magnetic resonance (NMR). Segmental motions do not occur and only the rapid rotation of the methyl groups of alanine residues is observed from ?120 to 130°C. This is independent of the conformation or form of the silk fibroin samples. Magnetization of dry silk fibroin by the solid-echo method shows a single Gaussian decay, while two components are observed in the solid-echo signals of films containing 6–10 w/w% water. An immobile component with a T₂ value of 11 μs is attributed to silk fibroin, and the mobile component to bound water. The T₂ of the latter varies from 50 to 200 μs, depending on the sample. The dynamical behavior of water trapped in the film is discussed on the basis of these T₂ values.     

Structural analysis of alanine tripeptide with antiparallel and parallel beta-sheet structures in relation to the analysis of mixed beta-sheet structures in Samia cynthia ricini silk protein fiber using solid-state NMR spectroscopy

The structural analysis of natural protein fibers with mixed parallel and antiparallel beta-sheet structures by solid-state NMR is reported. To obtain NMR parameters that can characterize these beta-sheet structures, (13)C solid-state NMR experiments were performed on two alanine tripeptide samples: one with 100% parallel beta-sheet structure and the other with 100% antiparallel beta-sheet structure. All (13)C resonances of the tripeptides could be assigned by a comparison of the methyl (13)C resonances of Ala(3) with different [3-(13)C]Ala labeling schemes and also by a series of RFDR (radio frequency driven recoupling) spectra observed by changing mixing times. Two (13)C resonances observed for each Ala residue could be assigned to two nonequivalent molecules per unit cell. Differences in the (13)C chemical shifts and (13)C spin-lattice relaxation times (T(1)) were observed between the two beta-sheet structures. Especially, about 3 times longer T(1) values were obtained for parallel beta-sheet structure as compared to those of antiparallel beta-sheet structure, which could be explicable by the difference in the hydrogen-bond networks of both structures. This very large difference in T(1) becomes a good measure to differentiate between parallel or antiparallel beta-sheet structures. These differences in the NMR parameters found for the tripeptides may be applied to assign the parallel and antiparallel beta-sheet (13)C resonances in the asymmetric and broad methyl spectra of [3-(13)C]Ala silk protein fiber of a wild silkworm, Samia cynthia ricini.     

Structure determination of a peptide model of the repeated helical domain in Samia cynthia ricini silk fibroin before spinning by a combination of advanced solid-state NMR methods

Fibrous proteins unlike globular proteins, contain repetitive amino acid sequences, giving rise to very regular secondary protein structures. Silk fibroin from a wild silkworm, Samia cynthia ricini, consists of about 100 repeats of alternating polyalanine (poly-Ala) regions of 12-13 residues in length and Gly-rich regions. In this paper, the precise structure of the model peptide, GGAGGGYGGDGG(A)(12)GGAGDGYGAG, which is a typical repeated sequence of the silk fibroin, was determined using a combination of three kinds of solid-state NMR studies; a quantitative use of (13)C CP/MAS NMR chemical shift with conformation-dependent (13)C chemical shift contour plots, 2D spin diffusion (13)C solid-state NMR under off magic angle spinning and rotational echo double resonance. The structure of the model peptide corresponding to the silk fibroin structure before spinning was determined. The torsion angles of the central Ala residue, Ala(19), in the poly-Ala region were determined to be (phi, psi) = (-59 degrees, -48 degrees ) which are values typically associated with alpha-helical structures. However, the torsion angles of the Gly(25) residue adjacent to the C-terminal side of the poly-Ala chain were determined to be (phi, psi) = (-66 degrees, -22 degrees ) and those of Gly(12) and Ala(13) residues at the N-terminal of the poly-Ala chain to be (phi, psi) = (-70 degrees, -30 degrees ). In addition, REDOR experiments indicate that the torsion angles of the two C-terminal Ala residues, Ala(23) and Ala(24), are (phi, psi) = (-66 degrees, -22 degrees ) and those of N-terminal two Ala residues, Ala(13) and Ala(14) are (phi, psi) = (-70 degrees, -30 degrees ). Thus, the local structure of N-terminal and C-terminal residues, and also the neighboring residues of alpha-helical poly-Ala chain in the model peptide is a more strongly wound structure than found in typical alpha-helix structures.     

Dissolution and regeneration of Bombyx mori silk fibroin using ionic liquids

In this work, the suitability of imidazolium-based ionic liquid solvents is investigated for the dissolution and regeneration of silkworm (Bombyx mori) silk. Within an ionic liquid the anion plays a larger role in dictating the ultimate solubility of the silk. The dissolution of the silk in the ionic liquid is confirmed using wide-angle X-ray scattering. The dissolved silk is also processed into 100 mum-thick, two-dimensional films, and the structure of these films is examined. The rinse solvent, acetonitrile or methanol, has a profound impact on both the topography of the films and the secondary structure of the silk protein. The image depicts a silkworm cocoon dissolved in 1-butyl-3-methylimidazolium chloride and then regenerated as a film with birefringence.     

Nucleation of hydroxyapatite crystals by self-assembled Bombyx mori silk fibroin

Bombyx mori silk fibroin (SF) is known to be capable of facilitating nucleation of the hydroxyapatite crystals (HAps). To find out how SF mediates the nucleation of HAps, self-assembly of SF in 1.5 simulated body fluid (SBF) was observed in this study through design of a co-solution of SF and 1.5-times SBF (SF/1.5 SBF). After the co-solution of SF/1.5 SBF was incubated at 37.2 °C up to 7 days, SEM, X-ray, and Fourier transform infrared (FTIR) observations indicated that nucleation of HAps was increased. In addition, the structure of SF was transited from random coil into β-sheet indicated by FTIR spectra. The β-sheet assembly of SF in 1.5 SBF was also supported by CD spectra. Atomic force microscopy provided detailed progress of the self-assembly that SF incubated in 1.5 SBF was self-assembled in the form from dot, through rod to final net. Therefore, this study suggested that nucleation of HAps of SF was controlled by its molecular self-assembly. © 2013 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys, 2013     

冬凌草甲素和冬凌草乙素与谷胱苷肽的迈克尔加成反应

采用紫外光谱动力学方法测定了抗肿瘤对映贝壳杉烯二萜冬凌草甲素和冬凌草乙素与谷胱苷肽迈克尔加成反应的级数、速率常数和平衡常数.结果表明,冬凌草甲素和冬凌草乙素与与谷胱苷肽迈克尔加成反应符合二级动力学方程,25℃下的速率常数分别为16.196 0L·(mol·s)-1和7.480 5L·(mol·s)-1,平衡常数分别为177.98L/mol和85.60L/mol.冬凌草甲素与谷胱苷肽迈克尔加成反应速率和反应程度均比冬凌草乙素的大得多,反应活性更好.对映贝壳杉烯二萜通过与机体发生迈克尔加成反应而产生抗肿瘤作用;因此,冬凌草甲素可能比冬凌草乙素具有更好的抗肿瘤活性.     

Solvent-free Michael addition reaction of fluorene with chalcon

A series of novel Michael addition products of fluorene to chalcone were obtained in the presence of sodium hydroxide under solvent-free condition.The advantages of this procedure were mild reaction conditions,simple protocol,and high yields.The structures of the products were characterized by IR,~1H NMR,MS and X-ray diffraction.The crystal of the new compound 3 h is Triclinic,space group P-1 with a = 0.97352(6) nm,b = 1.08918(7) nm,c = 2.58418(16) nm,α= 80.1400(10)°,β= 79.5490(10)°,γ= 64.2440(10)°,V = 2...     

Monte Carlo simulation of diepoxides and monoepoxides cured with amines

The diepoxide–monoepoxide–diamine curing systems are investigated with a Monte Carlo simulation. The dependence of the molecular weight distribution (MWD), gel fraction, and cycle rank of the polymers on the differences in the epoxy reactivities and the contents of the monoepoxide as a reactive diluent are discussed. Before gelation, the MWD of the curing systems with a lower content of the monoepoxide is broader than the MWD of the curing systems with a higher content, and it leads to a lower critical conversion. The gel fraction and cycle rank of the polymers decrease with an increasing amount of the diluent. Even fully cured, the system with a 0.6 epoxy molar fraction of the monoepoxide still has a large fraction of sol, about 49%. Although the various reactivities of the monoepoxide result in different ways of forming gels during curing, the final gel fractions are always near 100% as long as the epoxy molar fraction of the diluent is no more than 0.2. The profiles of the molecular weights of the polymers calculated by the model are in agreement with the experimental data. © 2002 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 40: 1857–1868, 2002     

Self-assembly of a peptide amphiphile based on hydrolysed Bombyx mori silk fibroin

An octapeptide, GAGAGAGY, was obtained by a novel method, i.e. hydrolysing Bombyx mori silk fibroin. Afterward, a dodecanoic acid-peptide conjugation was synthesized. This amphiphile assembled into cylindrical nanofibers of planar β-sheets at pH 9 and twisted β-sheets at pH 4.     

Defense role of the cocoon in the silk worm Bombyx mori L

Silk from the domesticated silk worm Bombyx mori procures foreign body response naturally, so it has been utilized as a biomaterial for decades. In India the prime focus of the sericulture industry is to improve silk production with high quality silk. Naturally, the silk worm builds its cocoon not only with silk proteins, but also with antimicrobial proteins to avoid infection since the cocoon is non-motile and non-feeding. The aim of the present study is to elucidate the antimicrobial proteins that persist in the cocoon of the silk worm Bombyx mori. At the pupal stage, the silk worm cocoon shell extract was prepared from the day of pupation (P0) to the day of natural rupture of the cocoon for the eclosion of moth (NR). Using the cocoon shell extract a microbial susceptibility test was performed by the disc diffusion method against the microbes Escherchia coli, Bacillus cereus, Staphylococcus aureus, Pseudomonas aeruginosa, and Klebsiella pneumoniae. The development of a zone of inhibition against the microbes confirmed the presence of antimicrobial/immunogenic activity of the cocoon shell extract. For further analysis, the cocoon shell extract was subjected to 7-15% sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE). The protein profile of the cocoon extract revealed the coomassie blue stained bands resolved from the 150-15 kDa molecular range. Interestingly, a polypeptide localized at around 29 kDa showed remarkable expressional changes during the development of pupa. To characterize the 29 kDa protein, it was eluted from the gel, digested with trypsin and analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The trypsin-digested peptide peaks were analyzed through MASCOT and peptides were matched with the NCBI nr database. The peptides were very well matched with the 18 wheeler protein, which is reported to be responsible for innate immunity, belonging to the Toll family in insects and responsible for cellular mediated immunity.     

用桑蚕丝素蛋白固定脲酶的研究及脲酶电极的制备

提出了一种新的酶固定化方法, 即通过甲醇处理, 使蚕丝素蛋白膜的构象由random coil向β-sheet发生根本性的变化, 从而将酶固定在β-sheet所特有的分子间氢键中。利用此方法所制成的脲酶电极, 在合适的操作条件下, 各项响应指标均令人满意, 并且脲酶的耐温性能被大大提高, 电极的有效使用寿命长达三个月以上。此种酶固定化方法原则上能够应用于其他不破坏蚕丝素蛋白分子结构的可溶性酶。     

Microstructure elucidation of historic silk (Bombyx mori) by nuclear magnetic resonance

¹H NMR cryoporometry and solid-state ¹³C cross-polarization (CP) magic-angle spinning (MAS) NMR spectroscopy were used to characterize the microstructure of historic and fresh silk samples. Silk is a polymeric bicomponent material composed of fibroin and water located in micropores. According to the ¹H NMR cryoporometry method, the intensity of the water resonance as a function of the temperature was used to obtain the pore size distribution, which was strongly asymmetric with a well-defined maximum at 1.1 nm. Compared with the fresh silk samples, the volume of pores around 1.1 nm decreased distinctly in the historic silk, and more pores larger than 2 nm emerged accordingly. In addition, these results correlated well with solid-state ¹³C CP/MAS NMR spectroscopy as the percentage of random coil in the historic silk sample was much less than that in the fresh silk samples. Therefore, it is suggested that the water-filled microvoids grow larger as the random coil conformation fades away in the degradation process.