Biomacromolecules 2002, 3, 644-648
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Articles Rheological Characterization of Nephila Spidroin Solution Xin Chen,*,†,‡ David P. Knight,† and Fritz Vollrath† Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS, U.K., and Department of Macromolecular Science, The Key Laboratory of Molecular Engineering of Polymers, Fudan University, Shanghai, 200433, People’s Republic of China Received September 20, 2001; Revised Manuscript Received April 17, 2002
We report the results of an investigation into the rheology of solutions of natural spider silk dope (spinning solution). We demonstrate that dilute dope solutions showed only shear thinning as the shear rate increased while more concentrated solutions showed an initial shear thinning followed by a shear thickening and a subsequent decline in viscosity. The critical shear rate for shear thickening depended on dope concentration and was very low in concentrated solutions. This helps to explain how spiders are able to spin silk at very low draw rates and why they use a very concentrated dope solution. We also show that the optimum shear rate for shear thickening in moderately concentrated solutions occurred at pH 6.3 close to the observed pH at the distal end of the spider’s spinning duct. Finally, we report that the addition of K+ ions to dilute dope solutions produced a spontaneous formation of nanofibrils that subsequently aggregated and precipitated. This change was not seen after the addition of other common cations. Taken together, these observations support the hypothesis that the secretion of H+ and K+ by the spider’s duct together with moderate strain rates produced during spinning induce a phase separation in the silk dope in which the silk protein (spidroin) molecules are converted into insoluble nanofibrils. Introduction Remarkably, orb web spiders are capable of turning a liquid dope into an extremely tough dragline thread without the use of high temperatures and pressures or toxic solvents.1 The transformation from liquid to solid takes place largely within the lumen of an elongated spinning duct.2-4 It involves the transformation of compactly folded protein (spidroin) possibly containing about 30% R-helices, 40% of so-called “random coil”, and 30% β-turns5 into a more extended conformation with a preponderance of antiparallel β-sheets. (For review see Winkler and Kaplan’s article.6) Although little is known about the way in which this is brought about, it has been suggested that it involves a strain-induced phase separation in which a concentrated dope solution containing the spidroin separates into a solid protein-rich phase (the thread) surrounded by an aqueous protein-poor phase.7 That such a strain-induced phase separation does indeed occur is indicated by the observation that the force required to draw silk from the spider’s spinneret shows an initial marked increase with reeling speed.8 Two observations point to a similar strain-induced phase separation in the formation of silkworm (Bombyx) silk.9 First, water was seen to flow from the external opening when aqueous solutions of regenerated * To whom correspondence may be addressed. Current address: Department of Macromolecular Science, Fudan University. Tel: +86-21-65642866. Fax: +86-21-6564-0293. E-mail:
[email protected]. † Department of Zoology, University of Oxford. ‡ Department of Macromolecular Science, Fudan University.
silk were spun from a narrow glass tubular die. Second, vigorously stirring a clear solution of regenerated silk resulted in formation of insoluble silk protein. The suggestion that the secretion of hydrogen ions into the spinning duct assists in this phase separation in both spiders7,10 and in silkworms11 is based largely on cytochemical evidence for the existence of a highly active proton pump in both systems. Recently, evidence from cryogenic temperature scanning electron microscopy energy dispersive X-ray (cryo SEM EDX) analysis that sodium and chloride ions are removed from the silk dope and potassium ions secreted into it as it flows through the spinning duct lead to the suggestion that an increase in the potassium/sodium ratio assists the phase separation process.12 Accordingly, we decided to test the effect of potassium ions and pH change on spider silk dope solutions. We show that potassium ions induce a two-stage process resulting in insoluble aggregates of silk protein nanofibrils. We also show that a strain-induced phase separation does indeed occur in concentrated solutions of spider silk dope and in more dilute solutions exposed to a reduction in pH to an optimal value close to that measured in the spider’s spinning duct. We discuss the possible mechanism underlying these changes and their significance to the natural spinning of spider silk. Experimental Section Preparation of Concentrated Dope. The amino acid composition and polyacrylamide gel electrophoretic behavior
10.1021/bm0156126 CCC: $22.00 © 2002 American Chemical Society Published on Web 05/23/2002
Characterization of Nephila Spidroin Solution
of the highly viscous material removed from the lumen of the major ampullate gland of a Nephila spider has been shown to resemble that of the fully formed dragline thread.13 This indicates that no major structural proteins are added, removed, or drastically modified in the spider’s spinning duct and that the protein contents of the sac can be regarded as those of the natural spinning dope. We have therefore used the term “concentrated dope” as shorthand for the contents of the major ampullate sac. Final instar female Nephila senegalensis spiders were killed by crushing the cephalothorax and immediately dissected in spider Ringer solution14 adjusted to pH 8.2 with Trizma base. The ampulla (sac) of the major ampullate gland was transferred to fresh Ringer and the epithelium removed to expose the concentrated dope. Care was taken to avoid shearing while handling it, as it is highly strain-sensitive when concentrated (see below). The concentrated dope was gently blotted for 1 s on Kleenex paper and transferred to a tared plastic micro-centrifuge tube. Drying samples of this blotted material to constant weight showed that the concentrated dope has a solids content of 34 ( 2%, n ) 6, N ) 3. Determination of the total amino acid content of these dried samples using the standard protocol for an Applied Biosystems high-performance liquid chromatograph indicated that protein accounted for 80 ( 18%, n ) 6, N ) 3 of the dry weight of the concentrated dope. Thus the protein concentration in the concentrated dope was approximately 25-30% (w/v), considerably less than the value of 50% suggested elsewhere5 but still a remarkably high concentration of protein. Preparation of Diluted Dope Solutions. Samples of the concentrated dope prepared as above were transferred to tared micro-centrifuge tubes, and after reweighing, 100 µL of fresh deionized water were added per 5 mg of the blotted dope to give a dope concentration of 5 wt %. We use the designation “dope concentration” throughout in this paper because there is as yet no rapid and convenient way to measure accurately the concentration of protein in each batch of dope before carrying out rheological investigations as the highly anomalous amino acid composition of the protein precludes the use of dye binding assays. The spider dope completely dissolved when left overnight at room temperature (approximately 20 °C) with occasional slight rotation of the tube. Dissolution was much slower at 4 °C and was therefore not used in this investigation. All dope solutions were stored at 4 °C after dissolution and used within 3 days of preparation. To investigate the effect of dope concentration, various dilutions were prepared from the 5.0 wt % dope solution using fresh deionized water. To investigate the effect of pH, 9 volumes of 0.2 mol/L sodium phosphate buffer solution were added to 1 volume of 5.0 wt % dope solution to make a final dope concentration of 0.5 wt %. To investigate the effects of different salts, 1 volume of 3.0 mol/L NaCl, KCl, CaCl2, or MgCl2 solution was added to 9 volumes of 1.0 wt % dope solution to make a final dope concentration of 0.9 wt % and salt concentration of 0.3 mol/L. Rheometry. Rheological measurements were performed on a Bohlin CVO 120 high-resolution rheometer using 20 mm diameter parallel plates. The plate gap was set to 200
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Figure 1. The rheological behavior of spidroin solution with different dope concentration.
µm for the more viscous 5.0 wt % dope and to 100 µm when more dilute dope solutions were used. The shear rate was linearly increased without oscillation from 0.5 to 1000 s-1 over 1000 s. Temperature was controlled at 25.0 ( 0.1 °C. Transmission Electron Microscopy. Precipitates produced from spidroin solutions were negatively stained with 2% ammonium molybdate solution before, and 15 and 90 min after, the addition of KCl solution as described above. Freshly prepared Formvar-carbon coated grids were used. The transmission electron microscope was operated at 75 kV. Results and Discussion Influence of Concentration on the Rheology of Nephila Spidroin Solutions. Typical rheological curves illustrating the effect of dope concentration from 0.05 to 5.0 wt % are shown in Figure 1. A simple shear thinning was found when the dope concentration was less than 1.0 wt %. This is hardly surprising as shear thinning has been reported for dilute solutions of soy proteins and other colloids.15 However, when the dope concentration was greater than 1.0 wt %, the rheology of spidroin solution was different, showing a marked shear thickening followed by a sudden drop of viscosity as the shear rate increased. The observed shear thickening strongly suggests a shearinduced formation of intermolecular aggregates as in certain synthetic polymer systems.16,17 This was confirmed by the observation that after applying high shear rates, we found irregular fibers of insoluble material suspended in a fluid phase between the rheometer plates. The details of the process involved in this aggregation are as yet unknown. The observed decline in viscosity after shear thickening is likely to result from almost all of the spidroin forming a highly hydrophobic intermolecular β-sheet structure that then precipitates. We have taken the critical shear rate at which the viscosity reaches a maximum in the shear thickening event as a measure of the ease with which spidroin undergoes a structural transition rather than using the less-defined point of onset of shear thickening (see Figure 2). Figures 1 and 2 show that an increase in dope concentration leads to a decline in the critical shear rate for shear
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Figure 2. Effect of dope concentration on the shear rate at which the viscosity drops suddenly after shear thickening (see text).
Figure 3. The rheological behavior of spidroin in different pH buffer solutions (dope concentration 0.5 wt %).
Figure 4. Plot of pH against the shear rate at which the viscosity in shear thickening reached a maximum (dope concentration 0.5 wt %).
thickening. Thus a change in dope concentration from 1.0 to 5.0 wt % resulted in a fall in critical shear rate from 890 to about 230 s-1. This increase in shear sensitivity is thought to result from an increase in intermolecular interaction16,17 between the spidroin molecules as the concentration increases. Influence of pH on the Rheology of Nephila Spidroin Solution. Figures 3 and 4 show the effect of pH on the rheology of 0.5 wt % spidroin solutions. Although no
Chen et al.
detectable precipitation occurred after the addition of buffer over the pH range 6.1-7.2, slight precipitation was seen at pH 6.0. Shear thickening could be detected in almost all buffer solutions from pH 6.0 to pH 7.2. Figure 3 shows two typical rheological curves of spidroin at different pH values. At pH 6.8, it shows shear thinning with only a small shear thickening peak around 300 s-1. No significant formation of precipitate was seen between the plates after shearing at this pH. In contrast, at pH 6.3 the shear thickening peak was higher, occurred at a much lower shear rate compared to pH 6.8, and resulted in obvious precipitation in the solution. For pH 6.4 and 6.5 (not figured), the maximum viscosity appeared at an even lower shear rate than at pH 6.3 and the copious precipitate caused the rheometer to jam (there was a dramatic and irregular change in both shear rate and viscosity). Jamming also occurred at a pH g 7.1. Figure 4 is assembled from rheological data for different buffer solutions and shows the effect of pH on the shear rate at which the maximum shear thickening viscosity was observed. The greatest sensitivity to shear occurred at about pH 6.4, while the solution was least sensitive at about pH 6.8. Interestingly, these values correlate well with measurements of the pH of the dope at the distal end of the spinning duct (pH 6.3) and in the sac (pH 6.9); thus it seems that the silk dope is spun at a pH at which it is most sensitive to shear and stored at a pH at which it is least sensitive. Many proteins show a conformation change between pH 6 and 7. Evidence is accumulating18-22 that a conformation change with this pH range results from a change in the protonation of one or a few histidine residues. These normally have a pK of about 6.3, but this can increase by as much as 1 pH unit depending on their environment in the protein.18 A decrease in pH not infrequently results in a conformation change involving a decrease in R-structure and an increase of β-structure. Such a transition “flip” occurs in heat shock protein Hsp47 close to pH 6.3 (ref 23) just as in spidroin. Influence of Salts on Nephila Spidroin Solution. There was no significant change of rheology when Na+, Mg2+, or Ca2+ ions were added to the spidroin to give final salt concentrations of 0.3 mol/L and dope concentrations of 0.9 wt %. However, when K+ ions were added to give the same final concentration, the solution immediately became turbid. Turbidimetry indicated that after mixing, light scattering reached a maximum in 0.5-1.0 h at 20° C and then decreased exponentially.24 Trials of increasing concentrations of KCl showed that a final KCl concentration
