Quantitative Change in Disulfide Bonds and Microstructure Variation

Jan 16, 2017 - of Regenerated Wool Keratin from Various Ionic Liquids ... University of Chinese Academy of Sciences, 19 A Yuquan Road, Beijing 100049,...
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Quantitative Change in Disulfide Bonds and Microstructure Variation of Regenerated Wool Keratin from Various Ionic Liquids Zhenlei Zhang,†,‡ Yi Nie,*,† Qiansen Zhang,† Xue Liu,§ Wenhui Tu,†,‡ Xiangping Zhang,† and Suojiang Zhang*,†

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Beijing Key Laboratory of Ionic Liquids Clean Process, State Key Laboratory of Multiphase Complex Systems, Key Laboratory of Green Process and Engineering, Institute of Process Engineering, Chinese Academy of Sciences, 1 North Second Street, Zhongguancun, Beijing 100190, China ‡ School of Chemistry and Chemical Engineering, University of Chinese Academy of Sciences, 19 A Yuquan Road, Beijing 100049, China § College of Chemistry and Chemical Engineering, Qufu Normal University, 57 Jingxuan West Road, Shandong 273165, China ABSTRACT: Ionic liquids (ILs) have been employed as solvents for dissolving wool keratin which can be further regenerated and applied in high-value and biocompatible keratin-based materials. It is urgent to determine the changes in disulfide bonds and keratin microstructures in the dissolution process for designing efficient ILs. Herein, the breakage of disulfide bonds by ILs was verified for the first time using a model compound, oxidized glutathione. Furthermore, at least 65% of disulfide bonds in keratin should be cleaved in order to be dissolved in ILs. The keratin regenerated in a series of ILs, and variable conditions were also characterized by a combination of S-linked techniques. The results indicated that changing both anions and cations can lead to a dramatically different ability to cleave disulfide bonds, while the side chains of the cations have little effect on it. Also, the dissolution variables, such as temperature and time, led to different amounts of disulfide bonds remaining in the regenerated keratin. Finally, molecular dynamics (MD) simulation found that the distribution of ILs around cystine was linked with the ILs’ disulfide bond cleaving ability. KEYWORDS: Wool keratin, Ionic liquids, Disulfide bond network, Cleavage

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by disulfide bridges, determining their excellent stiffness.9 Furthermore, strong intermolecular disulfide bonds form between adjacent keratin strands through oxidation of sulfide groups with the wool growing and extending away from the follicle.10 Therefore, the presence of a highly cross-linked disulfide bonds network and hierarchical microstructure makes it resistant to most of solvents. In turn, it is the prerequisite for realizing the reuse of wool keratin to appropriately break this kind of disulfide bond network in wool. Traditionally, chemical methods are employed to cleave disulfide bonds with the use of a strong acid, alkali hydrolysis, or other harmful chemicals like thiols. Although these solvent systems are efficient to handle keratin, they suffer from environmental problems and sometimes result in severe degradation of keratin. Recently, ionic liquids (ILs) have been widely applied to extract keratin from biopolymers.11−15 Several research groups have attempted to regenerate keratin with a number of ILs such as 1-allyl-3-methylimidazolium chloride ([Amim]Cl), 1-butyl-3-methylimidazolium chloride

INTRODUCTION Nature bestows us with plenty of materials with outstanding properties and functionalities. Keratin is a naturally fibrous protein which exhibits excellent performances within wide temperature ranges and unfolds beyond high limits.1 As one of the toughest biopolymers in organisms, it is abundantly available in wool, feathers, hair, animal horns, and hooves and plays structural and protective roles.2 These properties have led to the remarkable development of keratin-based materials with applications in biomedical devices,3 degradable bioplastics,4 animal feedstock,5 protein fiber,6 and film.7 Therefore, it is fascinating to use keratin for designing structural and functional materials. Wool keratin has received much attention due to its large quantities in the form of nonspinnable and short-fiber waste from the textile industry.8 However, the complex disulfide networks in wool is an obstacle to extract keratin from them. Wool fibers mainly consist of a core cellular component surrounded by a 0.5-μm outer cuticle layer. The cuticle layer is rich in disulfide cross-links due to the high cysteine content. In depth, the cortical cells, composing the core component, are made up of macrofibrils organized by microfibrils embedding into matrix proteins. Both macrofibrils and microfibrils are fixed © 2017 American Chemical Society

Received: December 6, 2016 Revised: January 9, 2017 Published: January 16, 2017 2614

DOI: 10.1021/acssuschemeng.6b02963 ACS Sustainable Chem. Eng. 2017, 5, 2614−2622

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ACS Sustainable Chemistry & Engineering ([Bmim]Cl),6 [choline][thioglycolate],11 N,N-dimethylethanolammonium formate ([DMEA][HCOO]),13 and 1-ethyl-3methylimidazolium dimethylphosphate ([Emim]DMP).14 These ILs showed different solubility for keratin around 120−130 °C. Taking dissolution efficiency and mechanical properties of the regenerated keratin into consideration, an ideal IL should meet the demand of good solubility for keratin and partially preserve the disulfide network in keratin, which could guarantee the strength of the regenerated material. The properties of ILs can be tuned by changing the structures of cations or anions,16 which enables one to obtain optimal solvents for dissolution and regeneration of keratin. In principle, the combination of various cations and anions can generate up to 1018 kinds of ILs.17 However, the basic science involved with changes in the disulfide network and microstructure during keratin dissolution in ILs is still in its infancy, and this may hold back the efficient utilization of ILs in processing biopolymers. This study is designed to investigate the quantitative variation of disulfide cross-link networks in the regenerated keratin and further provide a perspective on how ILs impact the keratin microstructure. A model compound was used to confirm the cleavage of disulfide bonds by ILs for the first time, shedding new light on why ILs have efficient dissolving capacity for proteins. In addition, keratin, regenerated from a series of ILs, was studied with a series of S-linked analytical techniques. Some dissolution variables, including structures of cations and anions, time, and temperature, were also explored to investigate their influences on the physiochemical properties of the regenerated keratin. At the molecular level, molecular dynamics simulation demonstrated that the disulfide bond cleavage rate and dissolution efficiency may be closely linked with the distribution of ILs around cystine. Thus, we present here a number of observations that help us gain further insights into the cleavage of disulfide bonds and change in the structural properties of the regenerated keratin from ILs.

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Figure 1. Cation and anion structures of the ionic liquids used in this study. with fresh methanol several times. Measurement of molecular weight was achieved by Matrix-assisted laser desorption/ionization time-offlight mass spectrometry (MALDI-TOF-MS). Dissolution and Regeneration of Wool Keratin. Wool fibers were immersed in ILs at the ratio of 1:10 (w/w), and the solution was mechanically stirred under certain times and temperatures. All the dissolving experiments were carried out under the atmosphere of air. Excess ethanol was added to the wool keratin/IL solutions to precipitate keratin, which was then separated by centrifugation for 5 min at 10,000 rpm and washed thoroughly with fresh ethanol several times to remove any residual ILs. Finally, the regenerated keratin was dried under vacuum at 60 °C for 24 h. The sample was ground into small particles in an agate mortar before testing. The recovery percentage was the ratio of the weight of regenerated keratin to the weight of raw wool. Characterization of Regenerated Wool Keratin. Solid 13C Nuclear Magnetic Resonance Spectroscopy (Solid 13C NMR). The solid 13C NMR spectra was recorded with a 400 MHz WB solid-state NMR spectrometer using a 4 mm rotor with an RF field of 62.5 kHz, 90° pulse, 4.17 μs, and 101 W. X-ray Diffraction (XRD). An X-ray diffraction study was performed to analyze the crystallinity of the wool fiber and the regenerated wool keratin. The data were collected at room temperature using a Bruker D8 Focus with diffraction intensities at 45 kV. The 2θ range was from 5° to 50°, while the scan speed was 0.05 s−1. The crystallinity index (CI) was calculated using the L. Segal method as shown in eq 1

EXPERIMENTAL SECTION

Material Preparation. The goat wool keratin fibers used were supplied by Dingda Biotechnology Co., Ltd. (Henan Province, P. R. China). Besides screening with meshes of 60 specifications, there was no further treatment. 1-Allyl-3-methylimidazolium chloride ([Amim]Cl, 98%), 1-butyl-3-methylimidazolium chloride ([Bmim]Cl, 98%), 1ethyl-3-methylimidazolium chloride ([Emim]Cl, 98%), 1-methylimidazolium chloride ([Hmim]Cl, 98%), 1-butyl-3-methylimidazolium bromide ([Bmim]Br, 98%), 1-butyl-3-methylimidazolium dibutylphosphate ([Bmim]DBP, 98%), 1-butyl-3-methylimidazolium dihydrogen phosphate ([Bmim]H2PO4, 98%), 1-butylpyridinium chloride ([BPy]Cl, 98%), 1-butyl-3-methylimidazolium acetate ([Bmim]OAc, 98%), [Emim]OAc (98%), tetrabutylphosphonium chloride ([P4444]Cl, 98%), and tetrabutylammonium chloride ([N4444]Cl, 98%) were purchased from Henan Linzhou Keneng Co., Ltd. The other two kinds of ILs, 1-ethyl-3-methylimidazolium dimethylphosphate ([Emim]DMP) and 1-ethyl-3-methylimidazolium diethylphosphate ([Emim]DEP), were prepared in our laboratory according to the procedures described in the literature.18,19 ILs, either purchased or lab synthesized, were dried at 80 °C in vacuum for 24 h before use. Figure 1 summarizes the cation and anion structures of the ILs used in this paper. Regeneration and Analysis of the Model Compound. Oxidized glutathione (GSSG) was chosen as the model compound because it contains disulfide bonds. After dissolving GSSG in ILs at 120 °C, the regeneration of GSSG was achieved through the addition of methanol to an IL/GSSG solution at 0−10 °C. The precipitate was separated by centrifugation for 10 min at 10,000 rpm after washing

CI = (I 9 − I14)/I 9

(1)

where CI is the crystallinity index, I9 is the maximum intensity of crystal lattice diffraction with 2θ at around 9°, and I14 is the minimum 2615

DOI: 10.1021/acssuschemeng.6b02963 ACS Sustainable Chem. Eng. 2017, 5, 2614−2622

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ACS Sustainable Chemistry & Engineering diffraction intensity with 2θ at around 14°. Generally, samples with higher values of CI have higher crystallinity.20−22 Determination of Disulfide Bonds and Sulfhydryl Group. Quantification of disulfide bonds and the sulfhydryl group in wool fiber and the regenerated keratin was achieved by a solid-phase assay according to the work by Chan23 and Zhao.24 The principle of this method is to dissolve a 10 mg sample in 0.8 mL of buffer (buffer A for quantification of free sulfhydryl groups: 8 M urea, 1% SDS, 3 mM EDTA, 0.2 mM Tris-HCl, pH 8.0; buffer B for quantification of total sulfhydryl groups: 8 M urea, 1% SDS, 3 mM EDTA, 0.2 mM Tris-HCl, 0.1 M Na2SO3, pH 9.5) and make it react with two color reagents, respectively, with the release of a measurable yellow-colored product. Then, the supernatant was removed and diluted, and its absorbance was read at 412 nm. Further, the Beer−Lambert Law was used to calculate its concentration. During the procedure, Ellman’s color reagent, 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB), was used to react with the free sulfhydryl groups,25 and a color reagent, disodium 2-nitro-5-thiosulfobenzoate (NTSB2−), was applied to quantify the total sulfhydryl group content.26 The disulfide bond content was calculated as the difference between the free sulfhydryl group content and total sulfhydryl group content. The molar extinction coefficient used for calculation was 13,600 M−1 cm−1. Each experiment was repeated in triplicate. Amino Acid Composition. The sample was first hydrolyzed in hydrochloric acid (6 mol/L), then monitored at 338 nm with an Alliance (Agilent 1100) high performance liquid chromatograph (HPLC). Elemental Analysis of Samples. The sulfur content of wool keratin was measured by an Elementar CHNS analyzer model Vario EL cube (Vario EL, Elementar Analyzer system. GmbH, Hanau, Germany). System Setup and Molecular Dynamic (MD) Simulation. Two separate simulation systems ([Emim]DEP-cystine, [Bmim]Cl]-cystine) were built by using Packmol,27 with initial densities equal to experimental values at the respective temperatures and pressures of each ionic liquid. Cubic simulation boxes are constructed with lengths of approximately 35 Å, and there is one cystine in each system. MD simulations were performed using the GROMACS 4.6 package,28 utilizing the CHARMM36 force field with cMAP corrections29 for the cystine. Parameters for ionic liquids were obtained by analogy30,31 from the CHARMM General Force Field.32 All simulations were performed as an NPT ensemble at 1.0 atm and 310 K, and with a time step of 2 fs. Constant pressure was maintained using the Nosé− Hoover Langevin piston method,33,34 and constant temperature was maintained by Langevin dynamics with a damping coefficient of 0.5 ps−1 applied to all atoms. Electrostatic interactions were calculated using the particle mesh Ewald (PME) algorithm35 with a real-space cutoff of 1.2 nm. Energy minimizations were first performed to relieve unfavorable contacts, followed by equilibration steps of 30 ns in total to equilibrate the solvent environment. Subsequently, two production simulations were submitted to each 200 ns. Trajectories were saved every 10 ps for analysis.

Figure 2. MS spectra of GSSG and regenerated GSSG.

Figure 3. Disulfide bonds and free sulfhydryl groups content of wool keratin regenerated from different ILs (120 °C, 30 min).

detected during the dissolution process, indicating cleavage of the disulfide bonds. There was also the release of H2S during dissolution of wool keratin at 120 °C. These results suggest that −S−S− is reduced to H2S during the wool keratin dissolution process in ILs. Content of Disulfide Bonds and Sulfhydryl Groups of Wool Keratin Regenerated with Different ILs. The disulfide bond network makes an important contribution to the stable three-dimensional conformation of wool keratin. Similar to traditional solvents used for dissolving wool keratin, ILs can also cause damage to the disulfide bond network. The contents of the disulfide bonds and sulfhydryl groups of regenerated wool keratin are presented in Figure 3. It was clear that the ability to cleave the disulfide network varied using various ILs (Table 1). Wool keratin regenerated from imidazolium-based acetate had the lowest amount of disulfide bonds, over 90% of which was ruptured, suggesting that [Emim]OAc and [Bmim]OAc had a higher ability of cleaving disulfide bonds. In contrast with other ILs, the wool keratin maintained a fibrous state during the 30 min dissolution

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RESULTS AND DISCUSSION Breakage of Disulfide Bond in Keratin by ILs. To solubilize wool, it is necessary to partially cleave the disulfide bond in keratin. The cleavage of disulfide bonds can be achieved by reduction, oxidation, sulfitolysis, or oxidative sulfitolysis with traditional methods.36 However, no work on how the disulfide bonds change by ILs is reported up to now. In order to see if there is any cleavage of the disulfide bond and to investigate how the disulfide bond changes during the dissolution, a disulfide bond-containing model compound, oxidized glutathione, was dissolved in [Emim]DEP at 120 °C. According to the procedure discussed above, the compound regenerated from the above solution was characterized by MALDI to determine its molecular weight. As shown in Figure 2, the standard peak of GSSG at 613 disappeared after regeneration from the IL solution. In addition, H2S was 2616

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disulfide breaking percentage. In addition, the ILs capability of dissolving wool keratin is proportional to the ability of cleaving disulfide bonds. As is illustrated in Table 1, at least 65% of the disulfide bond in keratin should be cleaved to achieve the dissolution in ILs. This may make it part of the criteria for designing and screening suitable ILs to dissolve keratin-based biomass. Microstructure Change in Keratin Regenerated with Different ILs. The solid state NMR spectra were collected to obtain more information about secondary structure of keratin. 13 C CP MAS spectra of raw and regenerated keratin are displayed in Figure 4. The NMR spectra of the regenerated keratin were similar to that of raw wool, suggesting that the main structure of keratin is maintained. The asymmetric peaks between 170 and 174 ppm are ascribed to carbonyl carbons, while the peaks at 53 and 38 ppm are attributed to α-carbon and β-carbon, respectively. In addition, the low chemical shifts are related with the alkyl groups of the side chains.12,37 The secondary structure of keratin containing α-helix and β-sheet resulted in a slightly different chemical shift of CO in spectra. Thus, the CO peak between 160 and 190 ppm was further resolved to obtain the fraction of α-helix, β-sheet, and random coil conformations.37,38 Take raw wool as an example; its resolved CO peak resulted in two peaks. The α-helix structure was associated with the peak at 172.5 ppm, and the peak with a maximum at 169.2 ppm was ascribed to β-sheet and random coil conformations. For the keratin regenerated from other ILs solutions, the resolved results are described in Table 2. The percentage fraction of the α-helix structure of regenerated keratin from ILs decreases in the following order: [N4444]Cl > [Emim]DEP > [Bmim]OAc. This order is consistent with the ILs’ ability of cleaving the disulfide bond discussed above. Therefore, the breakage of disulfide bonds in keratin can dramatically influence its secondary structure distribution. Interestingly, there is an increase in the α-helix structure in keratin regenerated from [N4444]Cl, which is due to the poor ability of [N4444]Cl to dissolve wool keratin, acting just as a cleaning agent. Effect of Cations and Anions on Cleaving Ability for Disulfide Bonds. Anions Effect. Relatively minor changes in cations and anions can lead to dramatic changes in the physicochemical properties, which could further affect its solvation ability for wool keratin. Here, we investigate a series of ILs to study the effects of cations and anions on dissolving wool keratin. Table 3(a) summarizes the disulfide bond content of keratins regenerated from ILs based on the [Emim]+ and [Bmim]+ cations. Results indicated that the remaining disulfide bond content in keratin regenerated from [Bmim]+-based ILs followed the order of [OAc]− < Cl− < Br−< [DBP]− < [H2PO4]−. In other words, the order of the ability to cleave −S−S− followed [OAc]− > Cl− > Br− > [DBP]− > [H2PO4]−. For the ILs incorporating [Emim]+ cation, the order was [OAc]− > Cl− > [DEP]− > [DMP] −. Both [Bmim]OAc and [Emim]OAc showed the highest cleaving ability for −S−S−. Additionally, the content of sulfur in regenerated keratin was also examined (Table 3(b)). A similar trend was observed compared with the disulfide bond content in keratin. The highlevel consistency between the disulfide bond content and the S content reveals that the sulfur in keratin mainly exists in disulfide bond networks. Cations Effect. To compare the influence of different cations on the dissolution of wool keratin, ILs with Cl− anions were

Table 1. Disulfide Bond Breakage Percentage and Recovery Percentage of Regenerated Keratin from Different ILs

a

ILs

breakage percentage (%)

dissolution status

recovery percentage (%)

[Emim]OAc [Bmim]OAc [Emim]Cl [Emim]DEP [Amim]Cl [Bmim]Cl [Emim]DMP [Hmim]Cl [BPy]Cl [Bmim]Br [Bmim]DBP [P4444]Cl [N4444]Cl [Bmim]H2PO4

95.05 92.92 78.73 77.89 70.12 67.47 65.05 61.18 55.82 28.85 1.79 − − −

Da Da Da Da Da Da Da PDb PDb PDb PDb NDc NDc NDc

15.77 16.82 73.86 70.20 73.75 78.54 76.60 − − − − − − −

Dissolution. bPartial dissolution. cNo dissolution in 30 min.

Figure 4. 13C NMR spectra of wool keratin and resolved spectra of raw wool.

Table 2. Secondary Structure Assessment of Raw Wool and Regenerated Keratin peaks

chemical shift (ppm)

HW (ppm)

fraction (%)

raw wool α-helix 172.5 7.17 85.74 random coil+β-sheet 169.2 3.72 14.26 keratin regenerated from [N4444]Cl solution α-helix 172.5 7.12 88.76 random coil+β-sheet 168.9 3.42 11.24 keratin regenerated from [Emim]DEP solution α-helix 171.9 7.18 76.24 random coil+β-sheet 169.2 4.52 23.76 keratin regenerated from [Bmim]OAc solution α-helix 176.8 9.05 23.09 random coil+β-sheet 171.1 6.98 76.91 It is fitted with Gaussian function. The fitting error is ±5%

process in quaternary ammonium and phosphonium ILs. These two ILs have a weak dissolving ability for wool keratin, with a high disulfide bond content in the regenerated keratin. The disulfide bond breakage percentage is also found to be associated with the recovery percentage of the keratin. The wool keratins regenerated from imidazolium-based acetate had the lowest recovery percentage while having the highest 2617

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Table 3. Quantification of Disulfide Bonds (a,c) and S Percentage Content (b,d) in Regenerated Keratin from ILs with Fixed Cation or Anion

Figure 5. Change in disulfide bonds and free sulfhydryl groups (a) and sulfur and cystine content (b) of the regenerated wool keratin at different dissolution temperatures.

used for the dissolution of wool. Table 3(c) and (d) show the results of the disulfide bond and sulfur content, respectively. Similar trends were observed in both tables. Accordingly, the effectiveness of the cations in cleaving disulfide bonds was found to be [Emim]+ > [Amim]+ ≈ [Bmim]+ > [Hmim]+ > [BPy]+ > [P4444]+ > [N4444]+. Overall, in the case of having the same anion, imidazolium-based ILs showed a similar ability of cleaving disulfide bonds, whereas quaternary ammonium and phosphonium ILs have a weak cleaving ability for disulfide bonds. This might be due to the large structures of [N4444]+ and [P4444]+. In addition, the small difference between imidazolium-based ILs reveals that the effect of side chains on disulfide bond cleavage is not obvious. Influence of Dissolution Conditions on Content of Disulfide Bonds and Microstructure of Regenerated Keratin. Temperature Effect. According to the literature,39 the dissolution temperature has significant effects on both keratin solubility in ILs and physicochemical characteristics of the regenerated keratin. The disulfide bond content of keratin regenerated at different temperatures was investigated. On the

Table 4. Recovery Percentage of Keratin Regenerated at Different Dissolution Time and Temperature (IL: [Emim]DEP) dissolution temperature (°C)

dissolution time (min)

recovery percentage

80 90 100 110 120 130 140 120 120 120 120 120 120

120 120 120 120 120 120 120 30 60 90 120 150 180

no dissolution no dissolution no dissolution 48.51% 30.63% 15.89% 11.36% 70.20% 40.62% 32.52% 30.63% 22.79% 20.56%

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Figure 6. Effect of dissolution time on the content of disulfide bonds and free sulfhydryl groups (a) and cystine and sulfur (b) in regenerated keratin.

Figure 7. XRD patterns of raw wool and regenerated wool keratin at different temperatures (a) and times (b).

Figure 8. Crystallinity index (CI) of raw wool and regenerated wool keratin at different temperatures (a) and times (b).

basis of our previous study,14 [Emim]DEP was chosen to perform the experiment because of its good performance in dissolving wool keratin. Figure 5(a) illustrates the quantitative changes in the disulfide bonds and sulfhydryl group content at

different temperatures. In terms of disulfide bond content, a clear declining trend was observed with an increase in temperature, indicating that the breakage of disulfide bonds rose with temperature. Interestingly, the sulfhydryl group 2619

DOI: 10.1021/acssuschemeng.6b02963 ACS Sustainable Chem. Eng. 2017, 5, 2614−2622

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Figure 9. MD simulations results of [Emim]DEP+cystine and [Bmim]Cl+cysteine. Spatial distribution functions of the cations and anions around cystine in their respective simulations (a). Radial distribution function (RDF) between the center of mass of the cysteine and the center of mass of the cations and anions (b). Representative simulation snapshots of the cystine in [Emim]DEP and [Bmim]Cl (c) and (d). For clarity, all hydrogen atoms have been hidden.

content increased first, reaching the maximum value at 110 °C and then decreased. At temperatures below 110 °C, the content of the free sulfhydryl group increased because of a rising number of cleaved disulfide bonds. The drop in sulfhydryl group over 110 °C could be attributed to the fact that the sulfhydryl group had higher reaction activity, which means that it is easy to transform to other compounds. The typical property that differentiates keratins from other structural protein is the rich cystine and sulfur content.40 During the process of dissolving wool keratin, the loss of cystine and sulfur was found to increase with the temperature rising (Figure 5(b)). Furthermore, the cysteine residues, participating in disulfide bond formation, can dramatically impact the degree of cross-linking of the polypeptide chain. Correspondingly, loss of cystine and sulfur was in accordance with the change in secondary structure distribution. The ordered α-helix structure, organized as coiled coils, would also decrease with temperature, while the β-sheet and disordered keratin increased with the rise in temperature. In other words, a higher dissolving temperature can accelerate the rate of degradation, which is associated with the rupture of the αhelix structure and disulfide bond breakage. In addition, the recovery percentage of the keratin at different dissolution temperatures was investigated (Table 4). The recovery percentage decreased with rising temperature. This is in accordance with the change in disulfide bond content in the regenerated keratin discussed above. Therefore, the ILs that

have a high dissolution ability for wool keratin at relatively low temperatures would be better solvents. Time Effect. Changes in the dissolution time can also influence the content of the disulfide bonds (Figure 6(a)) and sulfur as well as the cystine level (Figure 6(b)). These S-linked properties of the regenerated keratin all decreased with time, which suggested that the level of degradation of regenerated keratin raised with time at 120 °C. Further, it is shown in Table 4 that the recovery percentage decreased with time increasing, consistent with the decline of the disulfide bond, cystine, and sulfur content. Therefore, in order to avoid further degradation of keratin, the keratin should be immediately removed from the ILs solution when the wool is totally dissolved. The crystallinity of wool keratin regenerated at different times and temperatures was also investigated, and the X-ray diffractions are shown in Figure 7. The α-helix structure is shown in peaks at 2θ = 9° and 17.8°, while the peaks at 9° and 19° are ascribed to the β-sheet structure.41 In comparison with raw wool, there was a decrease in the XRD pattern at 9° and an enlargement of the peak at about 19°, which was mainly caused by the disruption of the α-helix structure.11,12,42 For a quantitative description of the crystallinity variation, CI was calculated with equation mentioned above. Figure 8(a) shows that the CI of regenerated keratin decreased sharply with temperature. There was also a drop in CI in keratin regenerated at different times compared with raw wool; however, the effect of dissolution time on CI was relatively unobvious (Figure 8(b)). 2620

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ACS Sustainable Chemistry & Engineering Notes

Distribution of ILs Around Disulfide Bonds. To obtain further information into the nature of the differentiated performance of ILs on cleaving disulfide bonds, we have simulated a cystine in each of the solvents ([Emim]DEP and [Bmim]Cl). By 200 ns MD simulations, it was found that anions, both DEP− and Cl−, are easier to gather around the disulfide bond of cystine. This result is well reflected by spatial distribution functions (SDF) and radial distribution function (RDF) methods of cations and anions around cystine (Figure 9(a) and (b)). In addition, it is shown in Figure 9(a) that the distributions of [Bmim]+ and Cl− were thin and dissociated compared to those of [Emim]+ and DEP− (in accordance with the experimental result that [Bmim]Cl had a weaker ability of cleaving disulfide bonds than that of [Emim]DEP). Also, the distribution of ILs around cystine is the possible reason that led to the difference in the ILs’ performance in cleaving disulfide bonds. From the representative conformations of ionic liquids with cystine, it can be further found that the polar groups of cations or anions are more likely close to the disulfide bonds of cystine (Figure 9(c) and (d)). These results also indicate that the polar groups of ILs may play a more important role in inducing the cleavage of disulfide bonds in the wool keratin.

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CONCLUSIONS

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AUTHOR INFORMATION

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is supported financially by the National Natural Science Foundation of China (21576262), National Natural Science Fund for Distinguished Young Scholars (21425625), International Cooperation and Exchange of the National Natural Science Foundation of China (51561145020), and “Recruitment of Outstanding Technologist” of Chinese Academy of Sciences and CAS Province Cooperation Program (2014JZ0012).

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REFERENCES

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In summary, the cleavage of disulfide bonds in keratin by ILs was confirmed for the first time using a model compound. In particular, H2S was detected during the dissolution of wool in ILs due to the breakage of disulfide bonds, which suggested that −S−S− was reduced during the dissolution in ILs. To realize the full dissolution of keratin in ILs, over 65% of disulfide bond needed to be cleaved. ILs with high cleaving ability for disulfide bonds can cause collapse of the microstructure of keratin making it unsuitable for obtaining keratin with good mechanical performances. Optimal ILs should have moderate disulfide bond cleaving ability (70−80%) to avoid severe damage of the keratin microstructure. In terms of the effects of dissolution variables on keratin, various ILs showed different abilities to cleave disulfide bonds, which may be associated with the distribution of ILs around the cystine. Both cations and anions had significant influence on the remaining content of disulfide bonds, while side chains of the same cationic base exhibited much less impact. Therefore, dissolution variables should be finely restricted to avoid the breakdown of the polypeptide chain. Overall, the results of this study, in addition to representing a step toward an improved understanding of the fundamental aspects of the change in disulfide bonds and microstructure of keratin during dissolution in ILs, have significant implications on how to preserve disulfide networks at the greatest extent by finely manipulating dissolution conditions. However, more work is necessary to study the detailed disulfide bond transformation mechanism and to obtain designing methods for screening ILs with moderate disulfide bond cleaving ability for wool dissolution.

Corresponding Authors

*E-mail: [email protected] (Y. Nie). *E-mail: [email protected] (S. Zhang). ORCID

Suojiang Zhang: 0000-0002-9397-954X 2621

DOI: 10.1021/acssuschemeng.6b02963 ACS Sustainable Chem. Eng. 2017, 5, 2614−2622

Research Article

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DOI: 10.1021/acssuschemeng.6b02963 ACS Sustainable Chem. Eng. 2017, 5, 2614−2622