Fibroin Chains Research Articles

Regenerated Bombyx silk solutions studied with rheometry and FTIR

Several different solvent systems are commonly used to dissolve Bombyx mori silk fibroin to prepare regenerated silk membranes and fibers though differences in the behavior of these solvents have not been fully investigated. Here we compare the effects of four of these on the rheology of silk fibroin solutions and on protein secondary structure as revealed by FTIR spectroscopy of cast membranes. The results demonstrated that Ca(NO 3) 2–MeOH–H 2O and LiBr–EtOH–H 2O had the strongest solvation on the silk fibroin chains, which showed an almost constant viscosity (Newtonian behavior) over most of the shear rate range (0.1–500 s −1). In contrast, the 9.5 M aqueous LiBr appeared to have the weakest solvation with similar effects on the silk fibroin molecules to pure water as indicated by rheological behavior. It was also found that the silk fibroin membranes prepared using all four solvent system showed mainly random coil conformation with a small proportion of β-sheet by FTIR spectroscopy. We discuss the implications of these findings for the preparation of regenerated silk for different applications.

Silk fibroin: structural implications of a remarkable amino acid sequence.

The amino acid sequence of the heavy chain of Bombyx mori silk fibroin was derived from the gene sequence. The 5,263-residue (391-kDa) polypeptide chain comprises 12 low-complexity "crystalline" domains made up of Gly-X repeats and covering 94% of the sequence; X is Ala in 65%, Ser in 23%, and Tyr in 9% of the repeats. The remainder includes a nonrepetitive 151-residue header sequence, 11 nearly identical copies of a 43-residue spacer sequence, and a 58-residue C-terminal sequence. The header sequence is homologous to the N-terminal sequence of other fibroins with a completely different crystalline region. In Bombyx mori, each crystalline domain is made up of subdomains of approximately 70 residues, which in most cases begin with repeats of the GAGAGS hexapeptide and terminate with the GAAS tetrapeptide. Within the subdomains, the Gly-X alternance is strict, which strongly supports the classic Pauling-Corey model, in which beta-sheets pack on each other in alternating layers of Gly/Gly and X/X contacts. When fitting the actual sequence to that model, we propose that each subdomain forms a beta-strand and each crystalline domain a two-layered beta-sandwich, and we suggest that the beta-sheets may be parallel, rather than antiparallel, as has been assumed up to now.

Determination of the site of disulfide linkage between heavy and light chains of silk fibroin produced by Bombyx mori

The analysis of fibroin secretion-deficient ‘naked-pupa’ mutant silkworms has suggested that the disulfide linkage between heavy (H) and light (L) chains of fibroin, produced by the silkworm, Bombyx mori, is essential in its efficient large-scale secretion from the posterior silk gland cells. However, the site of disulfide-linkage between H- and L-chains has not been determined. In this study, cysteine residues involved in the single disulfide linkage between H- and L-chains were identified as the twentieth residue from the carboxyl terminus of H-chain (Cys-c20) and Cys-172 of L-chain by sequencing of genomic clones and peptide analysis. Furthermore, Cys-c4 (fourth residue from the carboxyl terminus) and Cys-c1 at the carboxyl terminus of H-chain were shown to form an intramolecular disulfide bond.

Dynamic and Static Light Scattering of Dilute Aqueous Solutions of Silk Fibroin Collected from Bombyx Mori Silkworms

Dynamic (DLS) and static light scattering (SLS) were carried out on dilute aqueous solutions of silk fibroin samples collected from the posterior division of Bombyx Mori silkworms. DLS data identified the presence of the fast and the slow diffusive modes with diffusion coefficients different by 1 order of magnitude. The hydrodynamic radii RH were calculated from the fast and the slow modes by the use of the Stokes−Einstein relation as 16 and 240 nm, respectively. The z-average root-mean-square radius of gyration (〈s2〉z)1/2 (≡Rg) and the weight-average molecular weight, Mw, were determined from the SLS experiment as Rg = 330 nm and Mw = 1.3 × 107, respectively. The fast and the slow diffusive modes were interpreted as being due to the translational diffusive motions of the single fibroin molecule and clusters of fibroin chains associated through ionic bonding between COO- ions of the fibroin molecules and Ca2+ ions remaining in the sample. The form factor of the cluster was calculated and compared with theoretical predictions for branched flexible polymers.

Orientation of silk III at the air-water interface

A threefold helical crystal structure of Bombyx mori silk fibroin has been observed in films prepared from aqueous silk fibroin solutions using the Langmuir–Blodgett (LB) technique. The films were studied using a combination of transmission electron microscopy and electron diffraction techniques. Films prepared at a surface pressure of 16.7 mN/m have a uniaxially oriented crystalline texture, with the helical axis oriented perpendicular to the plane of the LB film. Films obtained from the air-water interface without compression have a different orientation, with the helical axes lying roughly in the plane of the film. In both cases the d-spacings observed in electron diffraction are the same and match a threefold helical model crystal structure, silk III, described in previous publications. Differences in the relative intensities of the observed reflections in both types of oriented samples, as compared to unoriented samples, allows estimations of orientation distributions and the calculations of orientation parameters. The orientation of the fibroin chain axis in the plane of the interfacial film for uncompressed samples is consistent with the amphiphilic behavior previously postulated to drive the formation of the threefold helical silk III conformation.

Bombyx mori silk fibroin liquid crystallinity and crystallization at aqueous fibroin–organic solvent interfaces

A banded morphology has been observed for Bombyx mori silk fibroin films obtained from an aqueous–hexane interface; the period of the banding is approximately 1 μm. Morphology and diffraction from different regions of the banded structure suggest that it is a free surface formed by a cholesteric liquid crystal. Truncated hexagonal lamellar crystallites of B. mori silk fibroin have been observed in films formed in the surface excess layer of fibroin at the interface between aqueous fibroin and hexane or chloroform. Based on initial crystallographic evidence, a three-fold helical conformation has been ascribed to the fibroin chains within the crystals. The chain conformation and crystalline habit appear to be similar to the silk III structure previously observed at the air–water interface (Valluzzi R, Gido SP. Biopolymers 1997;42:705–717; Valluzzi R, Gido S, Zhang W, Muller W, Kaplan D. Macromolecules 1996;29:8606–8614) but the crystalline packing is different. Diffraction data obtained for the crystallites are similar to diffraction behavior for a collagen-like model peptide. Diffraction patterns obtained from crystallized regions of the banded morphology can be indexed using the same unit cell as the hexagonal lamellar crystallites. Surfactancy of fibroin and subsequent aggregation and mesophase formation may help to explain the liquid crystallinity reported for silk, which is long suspected to play a role in the biological silk spinning process (Valluzzi R, Gido SP. Biopolymers 1997;42:705–717; Willcox, P. J.; Gido, SP, Muller W, Kaplan DL. Macromolecules 1996;29:5106–5110; Magoshi J, Magoshi Y, Nakamura S. In: Kaplan D, Adams W, Farmer B, Viney C, editors, Mechanism of Fiber Formation of Silkworm. Washington, DC: American Chemical Society 1994:292–310; Magoshi J, Magoshi Y, Nakamura S. J Appl Polym Sci Appl Polym Symp 1985;41:187–204; Magoshi J, Magoshi Y, Nakamura S. Polym Commun 1985;26:309.).

Swelling and dissolution of silk fibroin (Bombyx mori) in N-methyl morpholine N-oxide

Bombyx mori silk fibers were dissolved in N-methyl morpholine N-oxide (MMNO), an organic cyclic amine oxide used for the solvent spinning of regenerated cellulosic fibers. The commercial MMNO monohydrate used in this study as a solvent for silk is a hygroscopic compound crystalline at room temperature, which becomes an active solvent after melting at 76°C. The degree of hydration of MMNO was checked by DSC measurements. The solvation power of MMNO towards silk fibroin drastically decreased at a water content ≥20–21% w/w. Dissolution of silk required both thermal and mechanical energy. The optimum temperature was 100°C. At lower temperatures dissolution proceeded very slowly. At higher temperatures, rapid depolymerization of silk fibroin occurred. The value of the Flory–Huggins interaction parameter χ for the MMNO–H2O–silk fibroin system was −8.5, suggesting that dissolution is a thermodynamically favored process. The extent of degradation of silk fibroin was assessed by measuring the intrinsic viscosity and determining the amino acid composition of silk after regeneration with an aqueous methanol solution, which was effective in removing the solvent and coagulating silk. Regenerated silk fibroin membranes were characterized by infrared spectroscopy, differential scanning calorimetry and scanning electron microscopy. The prevailing molecular conformation of silk fibroin chains was the β-sheet structure, as shown by the intense amide I-III bands at 1704, 1627, 1515, 1260, and 1230 cm−1. The value of the I1260/I1230 intensity ratio (crystallinity index) was 0.68, comparable to that of the fibers. The DSC thermogram was characteristic of a silk fibroin material with unoriented β-sheet crystalline structure, with an intense decomposition endotherm at 294°C. The SEM examination of fractured surfaces showed the presence of a dense microstructure with a very fine texture formed by densely packed roundish particles of about 100–200 nm diameter.

Structure and physical properties of silk fibroin/polyacrylamide blend films

This article deals with the characterization of blend films obtained by mixing silk fibroin (SF) and polyacrylamide (PAAm). The DSC curves of SF/PAAm blend films showed overlapping of the main thermal transitions characteristic of the individual polymers. The exothermic peak at 218°C, assigned to the β-sheet crystallization of silk fibroin, slightly shifted to a lower temperature by blending. The weight-retention properties (TG) of the blend films were intermediate between those of the two constituents. The TMA response was indicative of a higher thermal stability of the blend films, even at low PAAm content (≤25%), the final breaking occurring at about 300°C (100°C higher than pure SF film). The peak of dynamic loss modulus of silk fibroin at 193°C gradually shifted to lower temperature in the blend films, suggesting an enhancement of the molecular motion of the fibroin chains induced by the presence of PAAm. Changes in the NH stretching region of silk fibroin were detected by FTIR analysis of blend films. These are attributable to disturbance of the hydrogen bond pattern of silk fibroin and formation of new hydrogen bonds with PAAm. The values of strength and elongation at break of blend films slightly improved at 20–25% PAAm content. A sea–island structure was observed by examining the air surface of the blend films by scanning electron microscopy. © 1999 John Wiley & Sons, Inc. J Appl Polym Sci 71: 1563–1571, 1999

The dissolution and characterization of Bombyx mori silk fibroin in calcium nitrate‐methanol solution and the regeneration of films

Bombyx mori silk fibroin from the silkworm was found to be soluble in a calcium nitrate-methanol system. Fibroin dissolves in 75% w/v Ca(NO3)2/MeOH solution at a temperature of 67°C. The viscometric behavior of the fibroin-salt solution was analyzed and the fibroin's secondary structures were determined via 13C solution nmr. Fourier transform ir, solid state 13C-nmr, x-ray diffraction, differential scanning calorimetry, scanning electron microscopy (SEM), and polarizing microscopy were used to characterize regenerated films and fibers. A compositional phase diagram of fibroin in the salt solution was constructed. Viscosity data indicate that there is aggregation of fibroin chains within the salt solution. The extremely high value of intrinsic viscosity of 8.7 dL/g at 298 K may be due to aggregation. Aggregation may be caused by the complexing of calcium ions with the fibroin chains at their amide linkages. The energy required for viscous flow for the fibroin solution (ΔHvis = 9.03 kcal/mol) is greater than that of the solvent (ΔHvis = 7.01 kcal/mol). Chain entanglements may be hindering the free motion of chains thus increasing the energy required for the viscous flow. 13C-nmr shows that fibroin chains exist in two independent conformational environments. While most of the molecule is in a random coil conformation, there is evidence of some order within the chains of fibroin. In as-cast regenerated films, the fibroin chains are in a random coil/α-helix conformation with some β-sheet content. Crystallinity induced by immersion of thin films in methanol is evidenced via x-ray diffraction, which shows lattice spacings at 4.042 A. Thin films have a fibrillar morphology that is clearly shown under the SEM and the polarizing microscope. Fibers were hand pulled from the concentrated fibroin-salt solutions and coagulated with acetone and methanol. A microscopic analysis was done using the polarizer. © 1997 John Wiley & Sons, Inc. Biopoly 42: 61–74, 1997

The dissolution and characterization of Bombyx mori silk fibroin in calcium nitrate‐methanol solution and the regeneration of films

Bombyx mori silk fibroin from the silkworm was found to be soluble in a calcium nitrate-methanol system. Fibroin dissolves in 75% w/v Ca(NO3)2/MeOH solution at a temperature of 67°C. The viscometric behavior of the fibroin-salt solution was analyzed and the fibroin's secondary structures were determined via 13C solution nmr. Fourier transform ir, solid state 13C-nmr, x-ray diffraction, differential scanning calorimetry, scanning electron microscopy (SEM), and polarizing microscopy were used to characterize regenerated films and fibers. A compositional phase diagram of fibroin in the salt solution was constructed. Viscosity data indicate that there is aggregation of fibroin chains within the salt solution. The extremely high value of intrinsic viscosity of 8.7 dL/g at 298 K may be due to aggregation. Aggregation may be caused by the complexing of calcium ions with the fibroin chains at their amide linkages. The energy required for viscous flow for the fibroin solution (ΔHvis = 9.03 kcal/mol) is greater than that of the solvent (ΔHvis = 7.01 kcal/mol). Chain entanglements may be hindering the free motion of chains thus increasing the energy required for the viscous flow. 13C-nmr shows that fibroin chains exist in two independent conformational environments. While most of the molecule is in a random coil conformation, there is evidence of some order within the chains of fibroin. In as-cast regenerated films, the fibroin chains are in a random coil/α-helix conformation with some β-sheet content. Crystallinity induced by immersion of thin films in methanol is evidenced via x-ray diffraction, which shows lattice spacings at 4.042 Å. Thin films have a fibrillar morphology that is clearly shown under the SEM and the polarizing microscope. Fibers were hand pulled from the concentrated fibroin-salt solutions and coagulated with acetone and methanol. A microscopic analysis was done using the polarizer. © 1997 John Wiley & Sons, Inc. Biopoly 42: 61–74, 1997

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 α-β structural transition of the fibroin chains.

Structural changes and dyeability of silk fibroin fiber following shrinkage in neutral salt solution

AbstractThe fine structural changes of Bombyx mori silk fibroin fibers, induced by shrinking with concentrated calcium chloride aqueous solution at elevated temperature were investigated as a function of shrinking rate. Tensile strength decreased and elongation at break increased in the shrinkage range 13–67%, the shape of the stress–strain curve changing from rubberlike to brittle at high shrinkage values (70–90%). The birefringence gradually decreased over the entire shrinking range examined, the curve becoming steep as the shrinkage exceeded about 67%. The behavior of isotropic refractive index (niso) closely resembled that of birefringence (Δn) in the shrinking range 13–67%. Beyond shrinkage of 67%, the niso showed a tendency to increase, especially for the sample with 80% shrinkage. Dichroism measurements showed that the molecular orientation within the amorphous regions decreased sharply at the beginning of the shrinking treatment, within the range 0–13%, then attained a saturation at about 55%. The position and intensity of the major X‐ray diffraction peak at 20.5° remained essentially unchanged regardless of the shrinking treatment. The results of dyeing behavior showed that the saturation value attained by shrunk silk fibers was significantly larger than that of the untreated control sample. Both standard affinity and the heat of dyeing increased slightly for the shrunk silk fibroin fiber, suggesting that a larger number of reactive sites became available for the interaction between dye molecules and fibroin chains. A schematic model is proposed for explaining the relation between structural changes and enhanced dyeability of the silk fibers following shrinkage in neutral salt solution. © 1994 John Wiley & Sons, Inc.

Changes in physical properties of methacrylonitrile (MAN)‐grafted silk fibers

AbstractChanges in physical properties of silk fibers, grafted with methacrylonitrile (MAN), were investigated as a function of the weight gain. The weight gain increased steadily during the first 60 min of reaction and gradually attained an equilibrium value (60%) after about 4 h. The initial tensile resistance of silk fibers decreased by MAN grafting. The crystalline structure of silk fibers remained unchanged, regardless of MAN grafting, however. a minor and broad peak appeared in the X‐ray diffraction curves of MAN‐grafted silk fibers with a weight gain of 60%, corresponding to the unoriented MAN polymer attached inside the fibers. Molecular orientation of silk fibroin chains in the crystalline regions, evaluated from X‐ray diffraction curves, did not change significantly, while both birefringence and isotropic refractive index decreased as the weight gain increased, implying that MAN polymer attached preferentially to the amorphous and not to the crystalline regions. Dynamic vis‐coelastic measurements showed that the position at which the E′ value began to decrease shifted to a lower temperature as the weight gain increased. These findings suggest that the thermal movement of silk fibroin molecules was accelerated by the presence of the poly‐MAN chains attached to the amorphous regions of silk fibroin fibers. © 1993 John Wiley & Sons, Inc.

Fibroinase from silk gland in the fourth molt stage in the silkworm, Bombyx mori

1. 1. High activity of fibroinase, an enzyme to hydrolyze the fibroin molecule, with complete hydrolysis of the heavy chain of fibroin within 1 hr in standard assay conditions, was demonstrated in extracted enzyme preparations from the silk gland in the fourth molt stage in the silkworm, Bombyx mori. Combined with cytological observations (Akai, 1965 Bull. Seric. exp. Sta. 19, 375–484) of the formation of vacuoles in the gland cells, followed by digestion of peripheral layers of fibroin and sericin in the lumen contents of the gland in the corresponding developmental stage, this result suggests that fibroinase is a secretory enzyme. 2. 2. Fibroinase in extracted enzyme preparation was subject to complete inhibition by antipain, leupeptin, chymostatin and E-64, all of which are potent protease inhibitors for cathepsins. This was the same result observed in the previous study on fibroinase from degenerating silk glands at day 1 (pharate adult). 3. 3. Low, but detectable, levels of fibroinase activity were observed in extracted enzyme preparations from silk glands at day 2 (fourth larval instar) and at day 0 (fifth larval instar). The results of the physiological function of fibroinase in the silkworm, Bombyx mori were discussed.

Occurrence of fibroinase in degenerating silk gland in the pharate adult of the silkworm, Bombyx mori

1. 1. The occurrence of an enzyme which hydrolyzes fibroin was demonstrated in degenerating silk gland in the pharate adult of the silkworm, Bombyx mori. The enzyme was named fibroinase. 2. 2. The assay system of fibroinase constructed in the present study consisted of tissue homogenate or its extract as an enzyme source, liquid fibroin prepared from solid fibroin as a substrate, and an identification system of hydrolysis of fibroin subunits, heavy and light chain fibroin, by use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 10% gels. 3. 3. Higher activity of fibroinase in tissue homogenate was observed at pH 5.0 with a complete hydrolysis of heavy chain fibroin after 5 hr of incubation. Dithiothreitol enhanced fibroinase activity. 4. 4. Fibroinase was extractable from the particulate fraction of tissue homogenate by the procedure used for extraction of cathepsins from lysosomes. Fibroinase in extracted enzyme preparation showed activity with a complete hydrolysis of heavy chain of fibroin within 20 min, 15-fold higher activity than tissue homogenate. 5. 5. Fibroinase in extracted enzyme preparation was subject to complete inhibition by antipain, leupeptin, chymostatin, and E-64, all of which are potent protease inhibitors for cathepsins.

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

AbstractThe 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 characteristics of silk fibers treated with epoxides

AbstractStructural characteristics of the silk fibroin fiber‐modified with epoxide treatment were elucidated by refractive index measurement, differential scanning calorimetry, and thermomechanical analysis and on the basis of strength–elongation measurements. The four epoxides used were glycidol (G), ethylene glycol diglycidyl ether (E), tolyl glycidyl ether (T), and resorcinol diglycidyl ether (R). The values of the isotropic refractive index of the silk fiber increased by the action of T or R. The position of the endothermic peak attributed to the thermal decomposition and the position of the onset of the contraction of each of the samples treated with epoxides shifted to higher temperature, suggesting the more thermal stability. However, the values of the molecular orientation and the crystallinity of the specimen evaluated from X‐ray diffraction analysis remained unchanged regardless of the epoxide treatment. These experimental results have been interpreted in terms of the cross‐links between the adjacent silk fibroin molecules and of the hydrogen bonds between the hydroxyl groups of the epoxide molecules attached to the side chains of the silk fibroin.

Effect of tRNA pool balance on rate and uniformity of elongation during translation of fibroin mRNA in a reticulocyte cell-free system

Unsuccessful attempts to synthesize complete fibroin chains in vitro were previously made in heterologous cell-free system [3]. In the present work, we succeeded to obtain complete translation of purified fibroin mRNA in a rabbit reticulocyte lysate. Whilst this work was being completed [1], similar results were published by Lizardi et al. [4]. The synthesis of full-sized molecules of fibroin (M.W. 360,000) was achieved by adding tRNA from the posterior silk gland to the cell-free system. With tRNA from other sources, both the translation rate and the amount of complete fibroin chains dropped. This effect of tRNA is situated at the elongation levels. Analysis of cell-free synthesized products by polyacrylamide gel electrophoresis shows that smaller discrete polypeptides are accumulated after 120 minutes of incubation. These polypeptides correspond to growing fibroin chains. This pattern of translation products suggests that elongation might decelerate at specific sites of the fibroin mRNA. These results show that a tRNA pool adjusted to mRNA codon frequency is required to obtain the maximal average elongation rate. A stochastic model based on random acceptance of tRNA at the ribosomal A site for the codon-anticodon recognition process can explain this phenomenon. It can also explain the occurrence of the unfinished discrete fibroin polypeptides during in vitro translation.

Discontinuous translation of silk fibroin in a reticulocyte cell-free system and in intact silk gland cells.

Silk fibroin mRNA was translated in a rabbit reticulocyte cell-free system. Addition of tRNA from silk glands was essential for complete translation of the fibroin polypeptide. (Mr approximately 400,000). Synthesis of full-sized product took at least 85 min. In addition to full-size product, a large number of smaller polypeptides were observed upon analysis by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Evidence is presented that these smaller polypeptides are growing fibroin chains that transiently accumulate as discrete size classes due to discontinuities in the translation process. These discontinuities, or pauses, occur at specific sites in the fibroin mRNA template. The relative duration of the pauses can be experimentally modulated by changing the source of the supplementary tRNA added to the in vitro system. Silk glands were incubated in organ culture under conditions where essentially exclusive labeling of newly synthesized fibroins was attained. Analysis in sodium dodecyl sulfate gels showed that the labeling pattern of nascent silk fibroins is similar to the pattern observed in the reticulocyte cell-free system. This result suggests that discontinuities or pauses in polypeptide chain elongation also occur in vivo under conditions of organ culture.

On the introduction of disulfide crosslinks into fibrous proteins and bovine serum albumin.

Hydroxyl groups in serine side chains of collagen, silk fibroin, and bovine serum albumin (BSA) were converted to SH by tosylation. In collagen film, 50% of the serine OH groups could be thiolated at most. In fibroin, only 13% because of its compact beta-pleated sheet structure and low susceptibility to swelling. The SH groups introduced are near enough together to form -S-S- bonds by oxidation. The residual SH content after oxidation was 0.1% in collagen and 0.03 to 0.25% in fibroin. Disulfide crosslinking increased the shrinkage temperature of collagen and fibroin and decreased the amount of shrinkage. BSA was crosslinked to dimers (MBSA) according to gel permeation chromatography and sedimentation analysis by the analytical centrifuge. Because these crosslinked proteins can be metabolized by the usual processes, in contrast to those crosslinked by artificial, nonphysiological bridges, they may be used for biological or medical purposes.