The chemical structure and the crystalline structures of Bombyx mori silk fibroin.

Some recent data (i.e. published in the last ten years) on the chemical and crystalline structures of B. mori silk are reviewed. The main emphasis is put on the crystallizable portion of silk fibroin, including its chemical constitution and its molecular conformation (at the crystallographic unit-cell level) in the two crystalline modifications : the beta pleated sheet and the silk I structures. The structural aspects are based on a discussion of X-ray and electron diffraction data, and on conformational energy analyses of a model (Ala-Gly)n polypeptide of silk fibroin.     

New process to form a silk fibroin porous 3-D structure

A new process to form fibroin spongy porous 3-D structure is reported herein. The process involves freezing and thawing fibroin aqueous solution in the presence of a small amount of an organic solvent. The process requires no freeze-drying, chemical cross-linking, or the aid of other polymeric materials. The solvent concentration, fibroin concentration, freezing temperature, and freezing duration affect the sponge formation, its porous structure, and its mechanical properties. Measurements by XRD and FTIR indicate that silk I and silk II crystalline structures exist in the fibroin sponge and that the secondary structure of fibroin is transformed to a beta-sheet from a random coil during this process. The tensile strength decreased slightly, but the fibroin sponge showed no deformation after autoclaving. Therefore, the fibroin sponge was sterilized using an autoclave. For 3 weeks, MC3T3 cells proliferated in the sterilized fibroin sponge. The fibroin sponge formed by this new process is applicable as a tissue-engineering scaffold because it is formed from biocompatible pure silk fibroin and offers both porous structure and mechanical properties that are suitable for cell growth and handling.     

Electron microscope examination of fibroin precipitated from silk fibroin solutions

The structure of gels formed from solutions of silk fibroin has been observed by electron microscopy. The gels consist of fibrils, and possible methods of formation of these fibrils are suggested.     

The crystal structure of Bombyx mori silk fibroin at the air–water interface

A new crystalline polymorph of Bombyx mori silk, which forms at the air–water interface, has been characterized. A previous study found this structure to be trigonal, and to be distinctly different than the two previously observed silk crystal structures, silk I and silk II. This new structure was named silk III. Identification of this new silk polymorph was based on evidence from transmission electron microscopy and electron diffraction, coupled with molecular modeling. In the current paper, additional data enables us to refine our model of the silk III structure. Some single crystal electron diffraction patterns indicate a deviation in symmetry away from a perfect trigonal unit cell to monoclinic unit cell. The detailed shape of the powder diffraction peaks also supports a monoclinic cell. The monoclinic crystal structure has an nonprimitive unit cell incorporating a slightly distorted hexagonal packing of silk molecular helices. The chains each assume a threefold helical conformation, resulting in a crystal structure similar to that observed for polyglycine II, but with some additional sheet-like packing features common to the threefold helical crystalline forms of many glycine-rich polypeptides. © 1997 John Wiley & Sons, Inc. Biopoly 42: 705–717, 1997     

An ESR study of spin-labeled silk fibroin membranes and spin-labeled glucose oxidase immobilized in silk fibroin membranes

This article describes the characteristics of silk fibroin membranes and glucose oxidase, immobilized in membranes as determined by a variety of physical methods, mainly the spin-label electron spin resonance (ESR) method. The properties of membranes insolubilized by different methods, i. e., immersion in 80% methanol aqueous solution, uniaxially drawing by placing on a stretcher, and hydration by placing in a desiccator of 96% relative humidity (RH) for 17 h, are compared. The results are also analyzed in relation to ESR spectra of spin-labeled immobilized glucose oxidase and 4-hydroxy-2,2,6,6-tetramethyl-1-piperidinyloxy as a model of the substrate. It is concluded that the heterogeneous structures of the swollen membranes in water differ locally among membranes insolubilized by different methods, but the immobilized state of the enzyme in such membranes is mostly similar. This is correlated to the fact that the thermal or pH stabilities are essentially same among glucose-oxidase-immobilized silk fibroin membranes insolubilized by different methods.     

The biosynthesis of fibroin. I. The intracellular form of silk fibroin of Bombyx mori L

After in vivo labeling with [³H]glycine the synthesis and transport of fibroin has been studied by radioautography and cell fractionation.Radioactivity appearing in the cytoplasm is rapidly transferred to the lumen where it accumulates in the so-called silk layer before reaching the central core of secreted fibroin. By sucrose density gradients it was demonstrated that the radioactivity appears immediately in the fibroin fraction, no precursors being observed.A simple fractionation procedure, based on the utilization of detergent, gives three fractions tentatively interpreted as synthesis, transport, and accumulation compartments in accordance with their kinetics of labeling.     

用桑蚕丝素蛋白制备邻苯二酚酶传感器

从蘑菇组织中提取邻苯二酚粗酶 ,利用丝素蛋白在甲醇作用下 ,其分子结构由可溶性randomcoil向不容性 β -sheet发生转变 ,从而将邻苯二酚粗酶固定在丝素蛋白膜中 ,制得邻苯一酚酶传感器。该传感器在pH6 0的KH2 PO4 -Na2 HPO4 工作介质中具有良好的响应特性 ,工作线性范围为 1 0× 10 - 5- 2 5× 10 - 4mol L ,检测限 5 0× 10 - 6 mol L ,响应时间 2min。酶经丝素蛋白的固定后具有较强的耐热性能 ,并能比较长时间保持酶的活性。该传感器在KH2 PO4 -Na2 HPO4 缓冲溶液的保存下 ,其使用寿命可达 2个月以上     

Phase behavior and hydration of silk fibroin

The osmotic stress method was applied to study the thermodynamics of supramolecular self-assembly phenomena in crystallizable segments of Bombyx mori silkworm silk fibroin. By controlling compositions and phases of silk fibroin solution, the method provided a means for the direct investigation of microscopic and thermodynamic details of these intermolecular interactions in aqueous media. It is apparent that as osmotic pressure increases, silk fibroin molecules are crowded together to form silk I structure and then with further increase in osmotic pressure become an antiparallel beta-sheet structure, silk II. A partial ternary phase diagram of water-silk fibroin-LiBr was constructed based on the results. The results provide quantitative evidence that the silk I structure must contain water of hydration. The enhanced control over structure and phase behavior using osmotic stress, as embodied in the phase diagram, could potentially be utilized to design a new route for water-based wet spinning of regenerated silk fibroin.     

Degradation mechanism and control of silk fibroin

Controlling the degradation process of silk is an important and interesting subject in the field of biomaterials. In the present study, silk fibroin films with different secondary conformations and nanostructures were used to study degradation behavior in buffered protease XIV solution. Different from previous studies, silk fibroin films with highest β-sheet content achieved the highest degradation rate in our research. A new degradation mechanism revealed that degradation behavior of silk fibroin was related to not only crystal content but also hydrophilic interaction and then crystal-noncrystal alternate nanostructures. First, hydrophilic blocks of silk fibroin were degraded. Then, hydrophobic crystal blocks that were formerly surrounded and immobilized by hydrophilic blocks became free particles and moved into solution. Therefore, on the basis of the mechanism, which enables the process to be more controllable and flexible, controlling the degradation behavior of silk fibroin without affecting other performances such as its mechanical or hydrophilic properties becomes feasible, and this would greatly expand the applications of silk as a biomedical material.     

A bacterial extracellular proteinase degrading silk fibroin

The bacterium Variovorax paradoxus, grown in a minimal medium in which silk fibroin represents the sole source of carbon and nitrogen, produces an extracellular protease that hydrolyzes fibroin as well as casein and, to a smaller extent, collagen and albumin. The optimal pH for activity was found to be in the acid range (optimum pH 5.8–6.4) and the enzyme activity was stimulated by the addition of divalent cations, either manganese or magnesium. Gel permeation chromatography and SDS-PAGE provided evidence that the native enzyme is a monomer with a M_r of ca. 21 kDa.