Dissolving Feather Keratin Using Sodium Sulfide for ... - Springer Link

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for Bio-Polymer Applications. Andrew J. Poole • Russell E. Lyons •. Jeffrey S. Church. Published online: 27 September 2011. Ó Her Majesty the Queen in Right ...
J Polym Environ (2011) 19:995–1004 DOI 10.1007/s10924-011-0365-6

ORIGINAL PAPER

Dissolving Feather Keratin Using Sodium Sulfide for Bio-Polymer Applications Andrew J. Poole • Russell E. Lyons Jeffrey S. Church



Published online: 27 September 2011 Ó Her Majesty the Queen in Right of Australia 2011

Abstract Feather keratin has been widely studied for use as a bio-based material. In this paper, we dissolve feather keratin using industrial sodium sulfide to investigate the yield, dissolved keratin characteristics, and properties of regenerated products to assess the potential of using sodium sulfide as a means of converting waste feathers into a biopolymer. Optimal conditions appeared to require short incubation times in order to give maximum strength in the regenerated product. This limits the yield to approximately 55%. Air-dried films and acid-precipitated samples are all readily re-crosslinked, suggesting the re-crosslinking process is robust. Minimizing exposure to the highly alkaline conditions appears favorable to final product strength through minimizing alkaline chain damage. The b-sheet structure of the parent keratin is largely maintained. The regenerated keratin was shown to have potentially attractive physical properties for use as a bio-polymer. Keywords Feather keratin  Dissolution  Sodium sulfide  Keratin films  Regenerated protein  Mechanical properties

Introduction Feather keratin has been widely studied for use as a biobased material [1–7]. It is a tough, high modulus, axially A. J. Poole  J. S. Church (&) CSIRO Materials Science and Engineering, PO Box 21, Belmont, VIC 3216, Australia e-mail: [email protected] R. E. Lyons CSIRO Livestock Industries, 306 Carmody Rd, St. Lucia, QLD 4067, Australia

oriented composite material [8, 9] of microfibrils within an amorphous matrix [10], containing b-sheet crystallites and is highly crosslinked by virtue of 7 mol% cysteine [11]. Primary chains are 10.2–10.4 kDa linear molecules containing a high number of hydrophobic residues [11, 12]. Feather keratin has mechanical strength similar to wool, and is likely the most abundant form of hard keratin in nature [7, 13]. An estimated 5 million tonnes of feathers are produced annually from a reliable production pipeline as a by-product of chicken meat farming [14], making it a dependable bio-polymer feedstock. Despite its properties, feather keratin is usually disposed of as waste or rendered into a low-grade feed stock [7]. One appealing end-use is to use the keratin as a natural monomer for use in eco-composites and bio-plastics [15]. For this, the challenge is to develop a technique for dissolving the keratin to produce solutions that are capable of re-crosslinking, that have minimal degradation of the primary protein chains, and that is industrially scalable. A wide number of techniques are available for dissolving hard keratin, often using reduction or oxidation reactions [16], and more recently, ionic liquids [17–19]. The use of 2-mercaptoethanol as a supplier of thiols is known to cleave the disulfide bonds without damaging the primary chain [20]. In an optimization of the 2-mercaptoethanol method, Schrooyen et al. [21–23] used a tenfold molar excess of 2-mercaptoethanol over the disulfide bond concentration in conjunction with 8 M urea, 3 mM EDTA, 200 mM Tris, pH 9, and 40 °C to achieve a yield up to 75% in 30 min. The urea acted to disrupt hydrogen bonding and swell the protein chains, giving better thiol ion penetration and an increased reaction rate. Reagents were removed by dialysis and re-crosslinking was prevented by the addition of sodium dodecyl sulfate (SDS) to the extracted keratins,

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with 100–200% additions (on weight of extracted keratin) giving SDS-keratin complexes comparable in size to the keratin primary chain [22]. The dissolved keratin was shown to readily re-crosslink in air to form solution cast films. Other workers added SDS to the dissolution mixture when dissolving feather or wool keratin [24–27] at approximately 85% (on weight of native keratin) to accelerate the extraction rate [24], although Schrooyen et al. [22] found no improvement from adding 140% SDS (on weight of native keratin). While the 2-mercaptoethanol method is a benchmark for good yield and undamaged keratin, the high cost of 2-mercaptoethanol means it is not industrially viable. A potentially simpler and much cheaper scheme using a single industrial chemical is to use sodium sulfide [28]. However, the reaction of sulfide with keratin is complex. The sodium sulfide reacts in water (Scheme 1) to form inorganic thiol (hydrosulfide) and hydroxyl ions [29], giving the reaction mixture a high pH. The hydrosulfide anion reduces the protein disulfide bond according to reaction Scheme 2. A mixture of the protonated and deprotonated forms of both of these highly reactive amino acid residues, cysteine and perthiocysteine, would be present. The effect of the high pH is twofold. It acts to disrupt hydrogen bonding and disaggregate the protein similar to urea in the 2-mercaptoethanol scheme. In addition, the hydroxide ions can reduce the disulfide bonds forming dehydroalanine by b-elimination (Scheme 3) [30]. The perthiocysteine residues produced by reaction Schemes 2 and 3 decompose to form cysteine and sulphur. Dehydroalanine residues are highly reactive and readily form cross-links with the amino acid side chains of cysteine and lysine to form lanthionine (Scheme 4) and lysinoalanine (Scheme 5). These reactions have the potential to provide a mechanism for cross-linking the proteins and thus potentially improving the physical properties of the end product. However, the strong reducing conditions generated by sodium sulfide have the potential to damage the protein backbone [31], although Jones and Meecham [28] supplied some evidence that they managed to cleave the crosslinks without causing substantial damage to the protein chain. A feather keratin dissolution scheme based on sodium sulfide could show promise for industrial application provided that sufficient yield can be obtained without alkalidamage to the primary chain. In this paper, we dissolve

Na2S + H2O

2Na+ + HS- + OH-

Scheme 1 The formation of hydrosulfide and hydroxide ion from sodium sulfide

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feather keratin using industrial grade sodium sulfide to investigate the yield, dissolved keratin characteristics, and properties of regenerated products to assess the potential of using sodium sulfide as a means of converting waste feathers into a bio-polymer.

Experimental Materials Feather Dissolution Feathers were collected fresh and still wet from a local chicken slaughterhouse. They were washed in a horizontal drum fitted with tines and rotating at 10 rpm. The washing process consisted of rinsing in water at 60 °C, washing in 0.4% aqueous Baymol, a non-ionic surfactant (Bayer, Australia), at 60 °C for 2 h, and then rinsing again at 60 °C. After draining, the feathers were sealed in cotton bags, spun to remove excess water using a commercial washing machine, and then dried in a commercial tumble drier at 70 °C for 3 h. The dried feathers were milled using a Wiley mill with 3.175 mm diameter die-plate holes. The milled feather was digested by placing 50 g feather in a 2 L conical flask with 500 mL of Na2S solution (technical grade, Ajax), filling the vessel headspace with N2 gas, and incubating at 130 rpm and 30 °C in a shaker incubator; Na2S concentration and incubation time were experimental variables. The mixture was then centrifuged at 1,0009 g for 10 min to isolate the supernatant from the un-dissolved solids. Additional dissolution experiments carried out included the use of sodium dodecyl sulfate (approx. 95% purity, Sigma) and urea (analytical grade, Ajax). A digestion run was also carried out in D2O (99.9 atom%, Aldrich). A reaction was carried out mimicking the digestion process in which elemental sulfur (precipitated, May & Baker Ltd., Dagenham, England) was used instead of feather keratin. Protein Films Films were cast from the supernatant in 140 mm diameter plastic Petri dishes that had been coated with a silicone based release agent. A heavy coating of the release agent was applied to the surface of the Petri dish, buffed with lint-free tissue until there was no visible trace of agent, washed with tap water, and buffed dry. A 40 mL aliquot of keratin solution was added to the Petri dish, which was placed on a level surface inside a fume hood and dried under a constant stream of fresh air at room temperature.

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R-CH2-S-S-CH2-R + HS-

R-CH2-SH + R-CH2-S-S-

Scheme 2 The reduction of disulfide bonds in cystine by hydrosulfide ion

C HC

O

NH

CH 2

S

S

CH 2 O

NH

OH -

+

CH C

C

O

C

CH 2

NH +

H2N

(CH2 ) 4

NH

O

CH C

cystine

lysine

dehydroalanine

C C

O

NH

CH 2

-

+

S

S

CH 2

CH

O

NH

dehydroalanine

C +

H 2O

C

NH

O

HC

CH 2

NH

(CH2 ) 4 CH

NH

O

C

perthiocysteine

lysinoalanine

Scheme 3 The formation of dehydroalanine by reduction of cystine

C

O

C

CH 2

+

HS

CH 2 O

NH

C HC

Characterization

CH

Protein Analysis

C

NH

O CH 2

NH

cysteine

dehydroalanine

S

CH 2

NH

Scheme 5 The formation of lysinoalanine by addition of lysine to dehydroalanine

O

CH C

lanthionine

Scheme 4 The formation of lanthionine by addition of cysteine to dehydroalanine

When dry, films were water insoluble. Residual Na2S was readily removed by soaking for 2 h in either tap water or in 1% HCl, as shown by testing for thiol with lead-acetate paper. Caution must be taken as lowering the pH has the potential to release H2S gas. For films cast with SDS, the residual detergent was removed by soaking overnight in 60% aqueous acetone saturated with KCl, adapted from the method of Lundgren [32]. Moisture content was measured from the wet state using standard conditions of 65% rh, 21 °C.

Protein concentration in the supernatant was determined spectrophotometrically at 280 nm (Shimadzu UV-2550) for samples diluted 1:100 with ultrapure water. A standard curve of absorbance as a function of protein concentration was determined gravimetrically by acid precipitating, water rinsing, and then drying samples of known absorbance overnight at 105 °C. It was found that an absorbance of 0.1 at 280 nm equated to 8.74 g L-1 of protein. SDS-PAGE samples were incubated at 95 °C for 10 min under reducing conditions (5% (v/v) 2-mercaptoethanol) and analyzed on 10% (w/v) Bis–Tris gels (Invitrogen). Gels were visualized with Coomassie Brilliant Blue solution (0.1% (w/v) and scanned using a Typhoon 9410 scanner (Amersham Biosciences). Data was processed using ImageQuant image analysis software, version 5.2 (Molecular Dynamics, Amersham Biosciences) to determine molecular weights of bands and their relative concentrations within each sample. Mechanical Testing Dog bone shaped test coupons, 4 mm wide in the test region, were cut using a knife press (Brooks Pneumatic Tool & Co, Melbourne, Vic) from films that were softened by soaking in water for 30 min. Ten coupons were cut from

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Raman Analysis Raman spectra were obtained at a resolution of 4 cm-1 using a RFS-100 FT-Raman spectrometer (Bruker) equipped with an Nd:YAG laser (Adlas) operating at 1,064 nm and a liquid nitrogen cooled Germanium diode detector. Raman data acquisition was performed using OPUS software v3.1 (Bruker). All data was collected using 180° backscatter geometry. Aqueous samples were held in a 5 mm path length quartz cuvette with a mirrored backing while precipitated and dried solids were packed into a 2 mm cavity cell. Spectra were obtained by co-adding 512 scans collected using a laser power of 750 mW. If there were no time restraints on data collection due to incubation, samples were analyzed in triplicate and averaged to produce the final spectra used for analysis. A Blackman–Harris 4-term apodization function was used. All spectral data manipulation was carried out using Grams AI software v7.02. For Raman analysis of the reaction mixtures, aliquots were taken from the digestion vessel and spun down to remove solids and thus stop the reaction. The clear supernatant was decanted and used for the analysis. For Raman analysis of solids precipitated from solubilized protein solutions the supernatant described above was termed Sample A. This supernatant was centrifuged again at higher force (10,0009 g for 10 min) and the highly clarified supernatant was termed Sample B and the pellet was termed Sample C. Aliquots of the Sample A supernatant were freeze-dried after shell-freezing. Additional aliquots were acid precipitated at pH 4 by drop-wise addition of 1% HCl with vigorous stirring. Again, caution must be taken as lowering the pH has the potential to release H2S gas.

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SEM Analysis Secondary electron images were obtained at low voltage using a Schottky emission variable pressure scanning electron microscope (Hitachi, Japan). Prior to imaging the samples were coated with 4 nm of chromium using a Dynavac Xenosput 2000.

Results and Discussion Feather Keratin Dissolution The cleaned and milled feather keratin was dissolved in aqueous sodium sulfide solutions ranging in concentration from 0 to 50 g L-1. As the results in Fig. 1 show, the reaction rate in the first 30 min was rapid and dependent on the Na2S concentration. After the first 1 h of incubation, there was no difference between the 10 and 50 g L-1 treatments, both concentrations giving a maximum dissolved keratin yield of 62% after 24 h. This value was only increased 3% by extending the incubation to 48 h (result not shown). This yield is lower than the 2-mercaptoethanol scheme [21] of around 75%, but is achieved with a significantly lower level of thiol, there being no advantage in going over a 3.5 fold excess—10 g L-1 Na2S providing 128 mmol thiol compared to the 36 mmol disulfide groups found in feathers [21]. By comparison, the 2-mercaptoethanol scheme used a tenfold excess. As previous workers had found the addition of urea and SDS to be beneficial [24], this was evaluated with dissolution solutions containing 10 g L-1 Na2S, 9 M urea and 10 g L-1 SDS. The results in Fig. 2 show that urea increased both the initial rate and the final yield. When added with urea, SDS had little effect after initially increasing the reaction rate, which is in agreement with Schrooyen’s [22, 23] findings that SDS gave no advantage after 1 h incubation time. When added without urea the 70

Na 2 S (g/L)

60

10 & 50

50

Yield (%)

each film; 5 were tested in the conditioned state (65% rh, 21 °C) and 5 were tested wet. For wet testing, the coupons were soaked for 1 h in ultrapure water at 21 °C and tested within 2 min of removal. Values reported are averages from duplicate films, although breakages due to brittleness reduced the number of coupons for testing in some instances. Coupons were stretched to breaking using an Instron 5500 (Instron, Switzerland) fitted with a 100 N load cell. The gauge length was 19 mm and the rate of extension was 5 mm min-1. Film thickness was measured using digital calipers, taking an average of 10 readings from along the length of the sample. Tensile strength, Young’s modulus and extension at break were calculated using Series IX Automated Materials Testing System software (Instron, version 8.10.00) and are reported as the means from the ten coupons tested. Statistical analysis (ANOVA and multiple comparison testing carried out at the 95% level of confidence) was carried out using Matlab (version R2010a, Mathworks) and the Statistics Toolbox.

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7.5

40

5

30

1

20 10

0 0 0

5

10

15

20

25

Solubilisation Time (h)

Fig. 1 Solubilization of feather keratin as a function of time for different Na2S concentrations at 30 °C

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65

Urea+SDS Urea

Yield (%)

60

SDS Control

55 50 45 40 35 0

2

4

6

8

Incubation Time (h)

Fig. 2 Effect of added SDS and urea on protein dissolution using 10 g/L sodium sulfide

Fig. 3 SDS-PAGE gel of dissolved keratin as a function of dissolution time using 10 g/L Na2S. Lanes are marked with the dissolution time in hours; lane M is the molecular weight marker standard. Numbers on the right represent distinct protein bands as detected by ImageQuant image analysis software

subtraction spectra obtained for the reaction are shown in Fig. 4. While there were no significant changes in the frequency of the features, significant spectral intensity changes are observed in the amide I (1,668 cm-1), S–H stretching (2,574 cm-1) and S–S stretching (449 cm-1) regions. In the representation used in Fig. 4, bands that become more negative with time represent species that are decreasing in concentration while those that increase in intensity with time are increasing in concentration in the solution. A

1003

Relative Intensity

reaction proceeded more quickly until 3 h incubation time, after which SDS had no effect on either rate or yield. The initial differences in yield % observed between the 10 g L-1 dissolution shown in Fig. 1 and the control dissolution shown in Fig. 2 reflects the inherent variability in the extraction process. All solutions containing Na2S in the first two experiments were found to have a pH of 14; the high pH disrupting hydrogen bonding to improve thiol access to the interior of the protein. The improved yield with urea shows access can be further improved beyond that achieved with high pH alone. The extent of damage caused by the high pH was assessed by analyzing the molecular weight fractions of the dissolved keratin as a function of reaction time using SDSPAGE. The results in Fig. 3 show that seven distinct protein bands were detected with little difference between times from 0.5 to 6 h. Longer treatment times show greater smearing of the gel, suggesting chain damage could be occurring. Gel quantification shows 68–70% of material is present as the 10 kDa fraction consistently across the time range investigated, and 15–19% is in the dimer form. By 24 h, the primary and dimer chains represent 98–99% of the material. While the heavy 10 kDa bands could have obscured fine differences, the consistency in molecular mass across the shorter times and a lack of fragments below the 10 kDa primary chain suggests the primary chain has remained relatively intact at the shorter treatment times. Raman spectra were obtained of aqueous keratin dissolved in 1% Na2S as a function of incubation time with treatments ranging from 0.5 to 29 h. The signal to noise ratio of these spectra, while sufficient for analysis, was limited as data collection time was restricted by the frequency that the reaction mixture was sampled. The spectral features attributable to water and the initial amount of dissolved Na2S were removed by subtraction of the spectrum obtained from the initial 1% Na2S solution. The Raman

Amide III Amide I

449 334

24

2574

6 4 0.5

2750

2250

1750

1250

750

250

-1

Wavenumbers (cm )

Fig. 4 The 2,750–250 cm-1 region of the Raman subtraction spectra obtained from the reaction mixture of milled feathers and 10 g/L Na2S at incubation times ranging from 0.5 to 24 h

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graph depicting these changes as a function of dissolution time is shown in Fig. 5. The amide I band intensity increases rapidly within the first hour of reaction, with no significant increases occurring after that time. This trend is consistent with that determined by UV analysis of the protein solutions (Fig. 1) and thus validates the method of Raman analysis. The amide I band peak maximum is observed at about 1,668 cm-1 with shoulders present at 1,660 and 1,680 cm-1. The frequency of 1,668 cm-1 suggests that the protein has remained largely in the b-sheet conformation [33]. The spectra of solubilized proteins have amide III band components at 1,250 and 1,238 cm-1, corresponding to random coil and b-sheet protein conformation, respectively [33]. The frequency overlap of b-sheet and random coil components in the amide I and III regions makes an accurate analysis very difficult. It appears however that both b-sheet and random coil protein conformation is present at all dissolution times. The thiol S–H stretching vibration is observed at 2,574 cm-1. The concentration of this species drops off with time in a somewhat linear fashion, no longer being detectable after 24 h. From comparison to the spectra obtained from the 1% sodium sulfide solution (not shown), the observed frequency can clearly be associated with that of the inorganic ion. For an aqueous solution of cysteine this vibration is observed at 2,578 cm-1. Under the conditions of the dissolution, this group is expected to be ionized and thus not expected to be observed. Disulfide bond cleavage is a key part of keratin dissolution. The extent of bond cleavage can be monitored using Raman spectroscopy as the S–S stretching vibration associated with cystine is observed as a well defined band complex near 512 cm-1 in the spectrum of the undigested feathers [34]. In the spectra obtained from the keratin protein solutions no clear evidence of this disulfide vibration is observed suggesting that a large portion of the disulfide bonds in the soluble protein have been cleaved.

% Change Peak Height

100

[Protein, amide I] 75

[-S-S- 449cm-1]

50

25

[-SH 2574cm-1] 0 0

5

10

15

20

25

30

Incubation Time (h)

Fig. 5 Graph of the % change in amide I, S–H stretching, S–S stretching peak intensities as a function of incubation time for the solubilization reaction

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As shown in Figs. 4 and 5, a spectral feature at 449 cm-1 appears and grows linearly in intensity as a function of dissolution time. Based on its frequency, strong intensity and sharpness, this band is likely associated with a S–S stretching vibration. Weaker features at 992 and 334 cm-1 also appears to be growing in with increased dissolution time. Bands at the same frequencies were observed when the digestion was carried out in D2O (results not shown) suggesting that the vibrations are not associated with protonated species. While the S–S stretching vibration of cystine is observed near 512 cm-1, the corresponding S–SO3 mode of cysteine-S-sulfonate is observed at 407 cm-1 [35]. The S–S and S–SO3 modes of cysteine-S-thiosulfonate are observed at 509 and 407 cm-1, respectively [36]. The possibility that the 449 cm-1 Raman line is associated with –S–SH or –S– S–SH species was tested by bubbling air through the dissolved protein solution. The S–H stretching vibration of the inorganic thiol anion observed at 2,574 cm-1 was found to decrease in intensity and disappeared but no changes were detected in the 449 cm-1 band intensity. It is possible that the 449 cm-1 band is associated with an inorganic species such as the thiosulfate ion which has strong S–S stretching modes at 452 and 433 cm-1 in the solid [36]. For thiosulfate, the A1 symmetric SO3 stretching vibration is observed at 1,016 and 991 cm-1 and the degenerate (E mode) S–S–O deformation is observed at 343 cm-1 [36]. Both of these Raman modes are moderate in intensity. The frequencies and intensities of these vibrational modes are consistent with those observed in the keratin dissolution time series spectra. As the digestions are carried out under N2, the formation of oxidized sulfur species would however be limited by the level of O2 dissolved in the water. Elemental sulfur, S8, exhibits a strong Raman band at 473 cm-1 with additional strong bands present at 217 and 152 cm-1 (spectrum not shown). While elemental sulfur is not soluble in water, polysulfide salts including sodium tetrasulfide are very soluble. Polysulfides are formed by the reaction of elemental sulfur and sodium sulfide, both of which are present during keratin dissolution. The compound Na2S4, which has been shown to exist in solution as a mixture of S42-, S52- and S2- species, exhibits a strong sharp Raman line near 446 cm-1 [37]. This line has been assigned to the stretching vibration of the central S–S bonds. The stretching modes of the external S–S bonds are observed as a weak shoulder at 482 cm-1. Considering the weak nature of this band and the high S/N of our spectra in this region it is not likely that this shoulder would be observed. When elemental sulfur was treated with sodium sulfide solution for 24 h, the Raman spectrum obtained from the clear supernatant exhibited a strong feature at 450 cm-1 as well as weak features at 997, 493 and

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338 cm-1. These spectral features are in excellent agreement with those observed in the spectra obtained from the dissolution time series, confirming the presence of polysulfides. Analysis of Isolated Protein Fractions In order to better understand the solubilization process, Raman spectra were obtained from protein solids obtained by acid-precipitating solutions after centrifuging the reaction mixture at 1,0009 g or 10,0009 g (Samples A and B, respectively) as well as the pellet (Sample C) of finely divided material obtained by re-centrifuging Sample A at 10,0009 g. Spectra obtained from the dried proteins isolated from a sample taken after 0.5 h of dissolution are presented in Fig. 6. For comparison purposes the spectrum from the milled feather keratin is also shown. In general, the spectra obtained from the two precipitated samples, A and B are very similar to that obtained from the milled feather sample (MF). Any spectral differences can be considered to be within the noise levels of the data. Significant changes however are evident when these spectra are compared to the spectrum obtained from the pellet, Sample C. The amide I and III regions of the spectra obtained from the precipitated fractions (A and B) are in very good agreement with those observed for the milled feather suggesting that the protein conformation is largely b-sheet. Unlike in the solution spectra (Fig. 4), the presence of protein disulfide bonds are clearly observed in the spectra obtained from the precipitated solids suggesting that these bonds have reformed. Based on disulfide peak areas, there is a 10–15% drop in bond density for the precipitated samples compared to the milled feather. For the pellet, a

S

Amide I

drop in peak area of the order of 25% can be estimated. A very notable and related difference observed in the spectrum obtained from the pellet is the presence of bands associated with elemental sulfur. No evidence of oxidized sulfur species can be detected in any of the Raman spectra collected from the samples in this series. The inorganic thiol anion band (region not shown) and the 449 cm-1 band, both of which are easily detected in spectra obtained from the solubilized protein solutions, are no longer present. This suggests that they are either being destroyed or removed during the precipitation and clean-up procedures. The major bands observed in spectra obtained from similar samples prepared from a 6 h feather keratin digestion were found to be in good agreement with those observed for the corresponding 0.5 h samples. Significant differences were however observed in the bands associated with functional groups involving sulfur atoms. The peak area of the disulfide bond stretching vibration has further decreased in the precipitated samples and there is a significant increase in the amount of elemental sulfur present in the pellet. The presence of sulfur in the spectra obtained from the pellets suggests that dehydroalanine is being formed according to reaction Scheme 1 and is immediately being destroyed in the formation of cross-links (Schemes 3 and 4). No spectroscopic evidence of the formation of these cross-links, increased secondary amine functionality in the case of lysinoalanine and increased C–S bonding (643 and 621 cm-1) for lanthionine formation, is detected as the change in band intensity would be small compared to that of similar functionality that is already present in the protein. The formation of these crosslinks, even in a minor amount, is however know to markedly decrease the solubility of fibrous proteins [30]. Considering the drop in number of disulfide bonds present in this fraction, the formation of lysinoalanine and lanthionine crosslinks are highly probable.

Relative Intensity

S

Feather Keratin Films C

-S-S-

B A MF 1700

1450

1200

950

700

450

200

Wavenumbers (cm-1)

Fig. 6 The 1,800–200 cm-1 region of the Raman spectra obtained from dried solids precipitated from a 0.5 h dissolution (10 g/L Na2S) after spinning for 10 min at 91,000 g (Sample A) and 910,000 g (Sample B) and the solid pellet obtained after 910,000 g (Sample C). The spectrum obtained from milled feather keratin is shown as trace MF

Mechanical properties such as tensile strength are important when considering a material for advanced applications. To this end, the effect of dissolution time was investigated. Keratin solutions were cast into films and air-dried to spontaneously re-crosslink. The resultant films were pale yellow and optically clear. An SEM image obtained from the surface of a typical film is shown as Fig. 7. The surface is homogeneous with a roughness on the 0.1 lm level. The small white sub-micron specks are debris from cutting the film. The moisture content of the films after conditioning was approximately 13%. As reported elsewhere [34], Raman analysis showed the films were isotropic with b-sheet conformation and a disulfide bond density similar to the

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Films 0.5 h

1,241(182)/ 121(39)

46(9)/8(2)

7(3)/8(3)

Films 1.0 h

1,568(271)/ 95(46)

61(7)/6(2)

7(2)/10(3)

digestion (95% confidence limit) or 1 h digestion with SDS (85% confidence limit). Measured in the wet state, the Young’s moduli of films from the 0.5 and 1 h digestions and the 1 h digestion with SDS were not statistically different. These samples did, however, have a statistically higher Young’s modulus than films cast from the 6 and 24 h digestions. No differences in mechanical properties were found between films before and after soaking to remove residual sulfide. The film produced from feathers digested for 1 h had superior mechanical properties to the best film reported by Schrooyen et al. [22] (Film 2-ME, Table 1). This was an optimized film that had 52% of the cysteine residues capped using iodoacetic acid and contained 0.15 g glycerol/g of keratin as plasticizer. Moore et al. [39] produced films without capped cysteine residues using an extraction method based on those of Schrooyen et al. [21] and Yamauchi et al. [24]. Their method incorporated 85% SDS (on weight of raw keratin) in the reaction mixture, a 1 h extraction time, and dialysis against distilled water to remove reagents (Film 2-ME ? SDS, Table 1). The Na2S films produced in the current study and those of Schrooyen et al. [22] were superior to the films of Moore et al. [39]. The film strength (conditioned state) of the 1 h digested feathers is similar to that of industrial polyester resin [40– 42], but is weaker than native feather keratin, particularly in the wet state, despite retaining b-sheet conformation and re-forming crosslinks to a similar density as the parent. This probably reflects the isotropic nature of the cast material compared to the parent which has longitudinally aligned microfibrils. Polymer drawing is known to increase strength [43], demonstrating that the elastic modulus of isotropic polyester is increased from 2 to just under 17 by drawing, which reduces chain entanglement while increasing chain alignment and crystallinity [44]. Stretching of the films cast from the 1 h digested keratin was shown to marginally increase alignment in the stretch direction [34], demonstrating alignment was possible.

Films 6 h

990(499)/ 23(10)

37(21)/3(1)

5(2)/17(4)

General Discussion

Films 24 h

NR/17(8)

NR/2(1)

NR/15(3)

Films 1.0 h with SDS

1,225(230)/ 73(43)

48(8)/5(1)

5(1)/15(6)

Optimal conditions appeared to require short incubation times in order to give maximum strength in the regenerated product. This limits the yield to approximately 55%. Even if incubation time is extended, yield is not significantly improved. It is not known what factors prevented maximum dissolution from progressing substantially further. Other workers also report maximum dissolution well below 100% for feather [21, 23] and wool [24, 31]. Using Na2S to solublise wool, Happey and Wormell [31] were limited to a yield of 65% despite using a much higher concentration and long time frame (250 g L-1 Na2S, 24 h at 25 °C). In contrast to feather keratin, wool proteins are heterogeneous

Fig. 7 An SEM image obtained from the surface of a typical film cast from a 1 h dissolution using 10 g/L Na2S and soaked to remove residual sulfide

parent material, similar to the Raman findings for the precipitated protein material presented above. In general, physical testing showed films with optimal mechanical properties were formed from short incubation times (Table 1). The Young’s modulus of the conditioned films from the 0.5 and 1 h digestions are not statistically different. However, film cast from the 1 h digestion has significantly higher Young’s modulus than films from either the 6 h

Table 1 Physical testing results of keratin films Sample

Young’s modulus (MPa) Conditioned/ wet

Tensile strength (MPa) Conditioned/ wet

Extension at break (%) Conditioned/ wet

Feather [38]

2,580/1,470

130/106

10.4/16.3

Films 2-ME [22]

1,344/–

30/–

3/–

Films 2-ME ? SDS [39]

10/–

17/–

2/–

Polyester resin [40–42]

4,000/–

45–85/–

2/–

SD are given in the brackets, 2-ME 2-mercaptoethanol, NR no result due to brittleness and fragility of samples, – no result reported

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with a generally higher molecular weight (10–55 kDa [45], higher cysteine content (10.5 mol% for whole merino fiber [46] with some sulfur-rich components containing 12–41 mol% [47]). These factors could be expected to cause some components to dissolve faster than others. Feather keratin protein is not only homogenous, but the precipitated dissolved protein and native keratins appear similar in structure based on the Raman analysis (Fig. 6), and an excess of inorganic thiol was still present even after the dissolution had appreciably slowed (Fig. 5). A contributing factor to limited dissolution may be the difficulty of the reductant gaining access to the interior of some larger keratin particles, as it was noticed the reaction mixture often contained undissolved particles of quill. Air-dried films and acid-precipitated samples are all readily re-crosslinked, suggesting the re-crosslinking process is robust. Raman analysis revealed disulfide levels were only 10–15% lower than the native material for short incubation (0.5 h) acid-precipitated samples, but samples with longer incubation (6 h) had decreasing disulfide content. Analysis of the finely divided particulate matter isolated from the reaction mixture (Sample C) showed 25% lower disulfide content than the bulk material as well as the presence of elemental sulfur, with the disulfide level further decreasing and elemental sulfur level increasing with longer incubation. Considering its low solubility and low disulfide crosslink content it can be postulated that this finely divided material is the result of re-crosslinking occurring via a dehydroalanine route to form lanthionine and/or lysinoalanine during the incubation. These crosslinking reactions are not prevented by the presence of reducing agent or high pH. The formation of these agglomerates is likely to be detrimental to the properties of a regenerated product as they were water-insoluble and so will not likely be crosslinked into the larger structure. Within the constraints of the SDS-PAGE method, alkaline chain damage did not appear to occur within the first 6 h of the incubation as there was no significant fragmentation of the primary chains. The loss in physical properties observed over this incubation period (Table 1) can therefore not likely be attributable to the degradation of the primary chains. An increased level of agglomerates in the films could however be one possible explanation for this drop-off. Therefore, minimizing exposure to the highly alkaline conditions appears favorable to final product strength.

Conclusions Sodium sulfide dissolved feather keratin in a simple scheme under ambient conditions, producing dissolved keratin that largely retains a b-sheet conformation and

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could be readily regenerated through air-oxidation. The quality of dissolved keratin was controlled via the dissolution time; with shorter times producing stronger regenerated products. At these reaction times, alkaline chain damage did not appear to be a significant problem. Regenerated keratin films had superior mechanical properties to films from a 2-mercaptoethanol digestion while using a simpler scheme without requirement for dialysis. The films demonstrated that the dissolved keratin had potentially attractive properties for use as a bio-polymer. Acknowledgments This work was funded by CSIRO Materials Science and Engineering. The technical assistance of Ms. Andrea Woodhead, Ms. Lisa O’Brien, Mr. Colin Veitch and Ms. Jacinta Poole is greatly appreciated.

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