Study on the effect of organic phosphonic compounds on disulfide bonds in wool

Abstract

In this study a new type of organic phosphonic compound named LKS-610, a kind of reducing agent, was used to cleave disulfide bonds in wool fibers. Research into the extraction of keratin from wool and processing into useful textile materials is a hot topic in the field of textiles. Wool fibers are rich in disulfide bonds which are the main chemical cross-linkage to maintain the fiber's secondary structure. If the disulfide bonds between molecular chains can be sufficiently disrupted, it will facilitate to obtain the keratin solution suitable for the regenerated materials preparation. The reaction mechanism and effect were analyzed through scanning electron microscopic test (SEM), X-ray photoelectron spectroscopy test (XPS) and amino acid content test. After a period of processing time, the valence state of sulfur element on fiber surface changed significantly; this result shows that the disulfide bonds reacted with LKS-610 reagent and generated thiol groups. Amino acid analysis demonstrated that for wool fibers treated for 60 min at 80℃, the quality percentage of cystine in wool was reduced from 10.09% to 1.05%, the majority of disulfide bonds had been cleaved. In this case, fiber scales were almost completely stripped and the cortical layer was not significantly damaged but became prone to swelling. This indicated that LKS-610 reagent could disrupt the disulfide bonds in the wool thoroughly and efficiently, and reduce these bonds to thiol groups. Wool fibers pretreated by LKS-610 reagent can be dissolved readily to prepare keratin solution; this keratin solution with high dissolution ratio and large molecular weight can be used in research into modification and preparation of regenerated protein materials.

Keywords

wool fiber reducing agent disulfide bond amino acid analysis

With the rapid development of science and technology, research into synthetic polymer materials has made significant achievements. However, most of the synthetic polymer materials are difficult to biodegrade, causing great damage to the environment. Therefore, research has been focused on the development and application of natural polymer materials. Each year, a large amount of wool fiber, rich in keratin, is abandoned, worldwide. If these fibers could be processed and utilized effectively, they would become valuable resources for regenerated protein polymer material.¹ At present, the study of wool recycled materials mainly is concentrated on two aspects: the dissolution methods of wool fiber^2,3 and the research into the performance of regenerated keratin.^4-6 The effective extraction of keratin from protein materials such as wool fibers is the basic premise for the study and production of regenerated protein materials (membrane or fiber). The preparation of the keratin solution plays an important part in processing. Methods that develop high dissolution ratio and keratin extract yield will avoid excessive waste of protein resources, thereby avoiding degradation of protein macromolecules during the dissolution process, and will facilitate production of regenerative keratin materials with excellent performance in future research work.

Wool consists of about 95% keratin proteins, which contain 10–15% cystine,^7,8 and cystine has an important role in determining the physicochemical properties of wool keratin. The disulfide bonds in cystine form a three-dimensionally linked network together with salt type bonds and hydrogen bonding.^9,10 It is well known that disulfide bonds have great bonding energy, making keratin's secondary structure difficult to damage easily,¹¹ so the wool fiber has higher stability and lower solubility. How to effectively break the disulfide bonds in the dissolving process while at the same time ensuring the protein macromolecule structure is not damaged or degraded too much is the key to obtaining high molecular weight keratin solution.

Commonly used methods for preparing wool keratin solutions are oxidation and reduction, but each method has some deficiencies. Oxidation has a higher dissolution ratio for wool fibers, and the commonly used reagents for this method are peroxides such as hydrogen peroxide and peracetic acid. However, the oxidative degradation of peptide chains is inevitable during the oxidation process. So, the average molecular weight of the keratin extracted by oxidation method is not high, it is generally distributed around 3 kDa–20 kDa.^4,12,13 The reduction method is widely used in current research on wool dissolution. This method uses a reducing agent to disrupt disulfide bonds between protein molecules to increase the solvency of wool fibers. The reduction method causes less damage to the protein, so the extracted keratin can maintain a larger molecular weight. The disadvantage of this method is that it is difficult for the reducing agent to fully react with the disulfide bonds in fibers, so the dissolution ratio and keratin extraction rate of the prepared keratin solution are always low (60 ∼ 70%),¹⁴ and the keratin resources in wool cannot be fully utilized.

In order to overcome the above problems, many new kinds of wool-dissolving methods and reagents have been reported in recent years. Liu et al. reported that the structures of ionic liquids had an important effect on their dissolution capability for wool keratin, and the 1-ethyl-1, 5-diazabicyclo[4.3.0]-non-5-enium diethylphosphate ([DBNE]DEP) can dissolved 8 wt% wool fibers in 3 h at 120℃.¹⁵ The keratin regenerated from [DBNE]DEP also exhibited high thermal stability and crystallinity. Wang et al. synthesized a series of ionic liquids and used these for the dissolution and regeneration of wool keratin. Phosphate-based ionic liquids were proved to have superior ability in dissolving wool fibers compared with acidic ionic liquids. Meanwhile, urea was chosen as the co-solvent in the process and showed good synergistic effects on the wool dissolution.¹⁶ Murate et al. found that molten urea could dissolve wool without the addition of water or any other chemicals. The molecular weight of water-soluble keratin was about 75 kDa, and the keratin was found to consist of 61% β-sheet and 39% α-helix. The water insoluble keratin consisted of only β-sheet.¹⁷

In this work, a new type of reductive reagent LKS-610 (organic phosphonic compounds) is used to disrupt the disulfide bonds in wool. This reagent's reduction potential is about –100 mw,so it has selective reduction for disulfide bond in proteins.¹⁸ Meanwhile, The LKS-610 aqueous solution has a pH value of about 8, which will not cause obvious alkaline hydrolysis of wool fibers in the treatment processing and it can prevent the protein chains from being disrupted excessively. The LKS-610 reagent can fully disrupt the disulfide bonds in a short period of time, and the wool fibers pretreated with this reagent can be dissolved more effectively. The chemical structure of LKS-610 reagent is shown in Figure 1 and the reaction mechanism of LKS-610 with disulfide bonds can be represented as the following:¹⁹

Figure 1.

Chemical structure of LKS-610 reagent.

The main objective of this work was to study the reaction mechanism and effect of LKS-610 reagent on disulfide bonds in fibers. The changes of surface morphology of wool fibers after treatment were examined by a field-emission scanning electron microscope test (FE-SEM). The valence state of the sulfur element in original fibers and treated fibers were compared using a X-ray photoelectron spectroscopy test (XPS). Meanwhile, the change of disulfide bonds quantity and other chemical structure changes in fibers after different treatment were analyzed by an amino acid content test.

Experimental

Materials

Cleaned wool fibers were supplied by Zhejiang Xinao Textile Inc. (China). LKS-610 (40%, analytical grade, pH = 8) was obtained from Tianjin Lvyuan Tianmei Technology Co., Ltd. (China). Thioglycolic acid (99%, analytical grade) and hydrochloric acid (37.5%, analytical grade) were purchased from Tianjin Kemiou Chemical Reagent Co., Ltd. (China). Lead acetate (analytical grade) was supplied by Tianjin Fengchuan Chemical Reagent Technologies Co., LTD. (China). Formic acid (88%, analytical grade) was acquired from Tianjin Kemiou Chemical Reagent Co., Ltd. (China) Coomassie Brilliant Blue R250 (analytical grade) was purchased from Nanjing Oddfoni Biological Technology Co., Ltd. (China).

LKS-610 treatment

The samples with 1 g clean dried wool fibers were cut up and treated with 10 mL LKS-610 reagent at 80℃ for 10 min, 30 min, 60 min and 90 min, separately. Then the samples were rinsed with deionized water, dried in air, and stored in sealed plastic bags for further processing.

Verification of disulfide bonds and thiols

The Ellman test has been always used to determine the content of thiols,^20,21 but this method could not be used in this study because the LKS-610 reagent can react with DTNB (5,5'-Dithiobis-(2-nitrobenzoic acid)). Other researchers have used lead ions reacting with sulfhydryl compounds to prepare nanoparticle or other insoluble substances,^22,23 the chemical reaction mechanism^24,25 represented as $\frac{2 R - SH + P {b^{2}}^{+} 60^{\circ} C}{\to RS - Pb - SR ↓ + 2 H^{+}}$ .

In this study, the LKS-610 reagent-treated wool fibers were soaked in lead acetate solution under a certain condition for reaction. SEM and XPS tests were conducted to determine whether sulfur and lead compounds could be formed on the surface of fibers. The presence of this insoluble demonstrated that the disulfide bonds were disrupted by the LKS-610 reagent and generated thiol groups. Meanwhile, lead ions reacting with thiol groups can prevent the thiols being oxidized to disulfide bonds in an aerobic environment, so the reaction effect of LKS-610 reagent on disulfide bond in wool can be studied more accurately by testing the cystine content changes.

The reaction products of thiols and lead ions were analyzed by the following method: 10 mL lead acetate solution was mixed with 10 mL LKS-610 reagent and 10 mL thioglycolic acid solution respectively. After 30 min of reaction at 60℃, the mixture was poured into culture dishes.

Specific method of reaction between fiber and lead acetate: the fiber samples were treated with LKS-610 reagent and immersed in 1.5 g/L lead acetate aqueous solution with ratio of 1:20 at 60℃ for 30 min. After the treatment, the samples were washed with distilled water several times to remove the impurities and unreacted lead ions adsorbed on the fiber surface, and then dried in preparation for the next test.

Scanning electron microscope (SEM)

The untreated and treated fiber samples were dried and sputter coated with gold and observed using a S4800 Scanning Electron Microscope (Hitachi, Japan) at 2000x magnification and at an accelerating voltage of 10 kV.

Optical microscope characterization

The fiber samples were fixed on a glass slide and observed under an optical microscope (Kyowa Optical Co., Ltd., Japan) and images were taken at 600×.

X-ray photoelectron spectroscopy test (XPS)

The X-ray photoelectron spectroscopy data of fiber samples were analyzed using an ESCALAB 250 photoelectron spectrometer (Thermo Electron VG Scientific, USA) with an MgKα (1253.6 eV) X-ray source, power was limited to 150 W to avoid degradation. The pressure of the chamber ranged from 10⁻⁷ Pa to 10⁻⁸ Pa, and the take-off angle was 45°. The curve fitting analysis was carried out using AugerScan 3.21 software (RBD Instruments, Inc., USA).

Amino acid analysis

Amino acid contents of wool fibers were tested using the L8900 amino acid analyzer (Hitachi, Japan). The samples were hydrolyzed in 6 M hydrochloric acid (HCL) for 24 h at 110℃ under nitrogen atmosphere. Free amino acid residues were derivative with hydroxyl succinimidyl carbamate (AQC, Waters, USA) and eluted on a reversed-phase column. An Alliance High Performance Liquid Chromatograph (HPLC) (Waters, USA) was used; the eluate was detected at 0.22 μm. The quantitative amino acid composition, expressed as mol% for each amino acid, was determined by external standard calibration (Amino Acid Standard H, Pierce).

Molecular weight distribution

Keratin solution was filtered and dialyzed using a dialysis bag (molecular weight cutoff 8000 Da) for 48 hours. The precipitated fraction obtained after dialysis was the keratin extracted from wool and the molecular weight of these keratin was determined by 12% SDS-PAGE using DYCZ-24EN vertical electrophoresis instrument (Beijing Liuyi Biotechnology, China). 3 μg keratin powder obtained after dialysis was added into a 7 μL sample buffer (mercaptoacetic acid and sodium dodecyl sulfate) and this mixed liquor was boiled in distilled water for 5 min to prepare the test protein sample. Protein separation was performed at 80 V for 120 min. After separation, gels were rinsed with ultrapure water for 5 min and staining was with Bio-Safe Comassie stain for 30 min. Destaining was done overnight in ultrapure water with gentle rotation and then the samples were compared with the protein molecular weight marker.

The treated fibers dissolved in formic acid solution

The LKS-610 reagent pre-treated fibers were immersed in 20 ml of formic acid solution and dissolved for a certain time at 60℃. After the dissolving process finished, the keratin solution was centrifuged and filtered to remove impurities that were not completely dissolved.

Results and discussion

SEM surface analysis

The fiber samples' SEM images after treatment by LKS-610 for different times are shown in Figure 2.

Figure 2.

SEM images of LKS-610-treated fiber samples. (a) untreated; (b) treated for 10 min; (c) treated for 30 min; (d) treated for 60 min; and (e) treated for 90 min.

The untreated wool fibers had complete scale layers, and the surface of the scales was smooth and flat (Figure 2(a)). When the fibers were treated with LKS-610 reagent for 10 mins (Figure 2(b)), part of the scales' edges peeled and the surface was no longer smooth. This was possibly due to reductive reagent LKS-610 reacting with disulfide bonds and causing the interaction between molecular chains to weaken. This made the wool scale layers soften, the surface was also etched and many grooves appeared.^26,27 Compared with the 10 min sample, the wool scales of the 30 min sample no longer retained an intact morphology. Figure 2(c) showed that most scales were damaged and fell off from the fiber. When wool fibers were treated for 60 minutes (Figure 2(d)), the fibers' scales layer was severely disrupted.²⁸ It could be seen that only a few incomplete scales remained on the fiber. After a further 90 mins, the scale layer was almost completely stripped and the fiber cortical layer's surface became very rough.

These changes in fiber morphology strongly suggested that LKS-610 reagent could effectively react with disulfide bonds in wool fibers and was able to strip the scale layer in a known processing time. Meanwhile, the fiber cortical layer was not damaged obviously but became susceptible to swelling.²⁹ This phenomenon illustrates that the wool fibers' scale layer is disrupted after LKS-610 reagent treatment and the fibers will no longer have good chemical inertness. So, by this means the pre-treated wool can be dissolved easily and efficiently.

The reaction of lead acetate and thiols

The treated fiber samples were examined by amino acid content test. The thiol groups, especially on the fiber surface, were easily oxidized to disulfide bonds again in the air or aqueous solution, which would affect the accuracy of cystine content test.³⁰ In order to avoid an oxidation effect, lead acetate was used to react with thiol groups and to generate insoluble substances. In this case, the thiol groups could be prevented from being oxidized into disulfide bonds again. The reaction results of thioglycolic acid and lead acetate are shown in Figure 3.

Figure 3.

The images of lead acetate solution and the reaction products of lead acetate with different reagents at 60℃. (a) lead acetate solution; (b) lead acetate with LKS-610 reagent; and (c) lead acetate with thioglycolic acid.

Figure 3(a) shows lead acetate aqueous solution at 60℃ for 30 min. A large amount of black precipitation could be found at the bottom of solution. This might be the result of lead oxide produced by the thermal decomposition of lead acetate in aqueous solutions. Figure 3(b) was the same state as Figure 3(a), with some black precipitates appearing at the bottom of the solution. This suggested that lead acetate would not react with LKS-610 reagent, so it could be used after wool fibers had been treated. Figure 3(c) showed the yellow precipitate generated when lead acetate reacted with thioglycolic acid; this kind of deposit is primarily the compound containing -S-Pb-S- bonds. The experiments proved that the thiol groups and lead ions could react under certain conditions and LKS-610 reagent would not affect their reaction.

In reference to the result above, untreated wool fibers and the fibers treated with LKS-610 reagent were immersed in lead acetate aqueous solution. Fibers were treated according to the processing conditions described in the section ‘Verification of disulfide bonds and thiols’; the treated fibers are shown in Figures 4 and 5.

Figure 4.

The images of fibers samples reacted with lead acetate. (a) untreated fibers; and (b) LKS-610 reagent treated fibers.

Figure 5.

The microphotographs of the fibers treated by LKS-610 reagents for: (a) 0 min; (b) 10 min; (c) 30 min; (d) 60 min; and (e) 90 min, and then reacted with lead acetate solution.

Two fiber samples' photographs were displayed in Figure 4. The sample in Figure 4(a) was light brown while the sample in Figure 4(b) was bright yellow. This was due to the untreated fibers not reacting in the lead acetate solution. However, there was a large number of thiols on the fiber surface after treated by LKS-610 reagent, and these thiols can react with lead ions to form yellow deposits on the fiber surface.

In Figure 5, the microphotographs of the fibers were collected to obtain more information about the reaction of wool thiols and lead ions. There was no significant change on the surface for untreated fibers (Figure 5(a)) and the fibers treated for 10 min (Figure 5(b)) soaked in lead acetate solution. In Figure 5(c), some deposits could be found on the surface of the fiber scales treated by LKS-610 reagent for 30 min. In Figure 5(d) and Figure 5(e) the fiber surface was almost covered with these deposits. The results indicated that a large number of disulfide bonds in wool were reduced by LKS-610 reagent and the fiber surface was rich in thiols after treatment. These thiol groups can combine with lead ions and generate yellow deposits.³¹ This conclusion will be further validated by the X-ray photoelectron spectroscopy test and amino acid composition test.

X-ray photoelectron spectroscopy (XPS)

The XPS spectra of the fibers soaked in lead acetate solution, the LKS-610 reagent treated fibers and the treated fiber samples blocked by lead acetate were compared with the untreated wool fibers. The sulfur element spectra of test samples is presented in Figure 6.

Figure 6.

Sulfur XPS spectra of (a) untreated wool fibers, (b) untreated fibers soaked in lead acetate solution, (c) fibers treated by LKS-610 reagent and reacted with lead acetate, and (d) LKS-610 reagent treated fibers.

It suggested that sulfur elements involved in the reaction with the LKS-610 reagent and its valence state changed.^32,33 The spectrum of the sample in Figure 6(a) can be fitted into two peaks: 164.08 eV and 165.16 eV. The peak at 164.08 eV corresponds to -S-S- bonds (164.0 eV).³⁴ Figure 6(b) shows the peaks of the sample's binding energy were similar to the sample in Figure 6(a), two peaks at 164.06 eV and 165.58 eV, respectively. When the wool fibers were soaked in a high temperature aqueous solution, some of the cysteine residues in wool would be hydrolyzed and oxidized. As seen in Figure 6(b), one peak (164.06 eV) corresponded to -S-S- bond, and the other (165.58 eV) to -S-O- bond.³⁵ It also proves again that untreated wool fibers would not react with lead acetate. The sample which reacted with LKS-610 reagent and then lead acetate is shown in Figure 6(c), whose binding energy was obviously different from that in Figure 6(a). The results showed that two peaks appeared at 161.60 eV and 162.88 eV respectively, while the peak at 164.0 eV disappeared. From the element binding energy table it could be found that the binding energy of sulfur elements in PbS and Pb(SR)2 was 161.9 eV and 162.8 eV respectively. The results suggested that the LKS-610 reagent cleaved the disulfide bonds in the wool fiber and then the thiol groups were produced. These thiol groups could combine with lead ions under certain conditions and generated insoluble matter. The sample in Figure 6(d) showed two peaks. One corresponded to the disulfide bonds (164.0 eV), and the other at 163.28 eV was close to the binding energy of the -SH (163.6 eV). It indicated that in air or water, the newly generated thiol groups were easily oxidized, of these one part was reoxidized to disulfide bonds and the other part remained in the form of thiol groups. The conclusions from the XPS test proved again that LKS-610 reagent would cleave the disulfide bonds in wool and generate thiols.

Amino acid composition of fiber samples

The amino acid compositions of the untreated wool fiber sample and LKS-610 reagent treated fiber samples were evaluated, as listed in Tables 1, 2, Figures 7 and 8, in terms of the mass percent of each kind of amino acids.

Table 1.

Amino acid compositions of different samples (1). Sample A: untreated wool fibers reacted with lead acetate for 30 min; Sample B: wool fibers treated by LKS-610 reagent for 30 min

	Untreated sample		Sample A		Sample B
	Mass (g)	Mass percent (%)	Mass (g)	Mass percent (%)	Mass (g)	Mass percent (%)
Aspartic acid	5.1642	6.26	4.7076	6.04	5.0873	7.23
Threonine	4.8305	5.85	4.4739	5.74	3.9901	5.67
Serine	7.0301	8.52	7.5826	9.72	5.7444	8.16
Glutamic acid	11.0744	13.42	11.7842	15.11	10.3003	14.63
Glycine	4.4074	5.34	4.2525	5.45	3.9170	5.56
Alanine	3.3423	4.05	3.0689	3.93	3.7902	5.38
Cystine	8.3250	10.09	7.8376	10.05	2.6758	3.80
Valine	3.9908	4.83	3.2504	4.17	3.7898	5.38
Methionine	0.6273	0.76	0.7906	1.01	0.7310	1.04
Isoleucine	2.6642	3.23	2.3243	2.98	2.2984	3.26
Leucine	6.9586	8.43	6.5273	8.37	6.1518	8.74
Tyrosine	4.2573	5.16	4.3230	5.54	3.9380	5.59
Phenylalanine	3.1736	3.84	2.9413	3.77	2.7530	3.91
Lysine	2.8658	3.47	2.6716	3.43	2.7008	3.84
NH₃	1.2417	1.50	0.6529	0.84	1.1092	1.58
Histidine	0.9417	1.14	0.8990	1.15	0.8353	1.19
Arginine	7.8702	9.53	7.3904	9.48	7.1122	10.10
Proline	3.7755	4.57	2.5191	3.23	3.4837	4.95
Total amino acid content	82.5453	100	77.9979	100	70.4084	100

Table 2.

Amino acid compositions of different samples (2). Wool fibers treated by LKS-610 reagent for: 10 min (sample C); 30 min (sample D); 60 min (sample E); 90 min (sample F) and then reacted with lead acetate for 30 min

	Sample C		Sample D		Sample E		Sample F
	Mass (g)	Mass percent (%)	Mass (g)	Mass percent (%)	Mass (g)	Mass percent (%)	Mass (g)	Mass percent (%)
Aspartic acid	4.9143	6.95	5.4272	7.46	5.2838	7.59	3.2196	5.99
Threonine	3.9183	5.54	4.1745	5.74	3.8973	5.60	3.1405	5.85
Serine	6.5463	9.25	5.9702	8.21	5.6030	8.05	5.6494	10.53
Glutamic acid	10.0535	14.21	11.2090	15.42	10.8190	15.54	9.3532	17.43
Glycine	3.8759	5.48	3.8961	5.36	3.6453	5.24	2.9764	5.55
Alanine	3.5272	4.98	4.4742	6.15	4.6736	6.71	3.4729	6.47
Cystine	2.4445	3.45	1.1237	1.55	0.7323	1.05	0.5531	1.03
Valine	3.8293	5.41	3.7011	5.09	3.4408	4.94	2.3292	4.34
Methionine	0.6695	0.95	0.4971	0.68	0.4156	0.60	0.4647	0.86
Isoleucine	2.2513	3.18	2.5524	3.51	2.4840	3.57	1.7399	3.24
Leucine	6.1324	8.67	7.0161	9.65	6.9078	9.92	5.3954	10.05
Tyrosine	4.1693	5.89	3.7484	5.16	3.5724	5.13	3.0659	5.71
Phenylalanine	3.1040	4.39	2.6047	3.58	2.6310	3.78	2.1914	4.08
Lysine	2.8444	4.02	2.9265	4.03	2.9570	4.25	2.3807	4.44
NH₃	1.2093	1.71	1.1795	1.62	1.1305	1.62	0.5737	1.07
Histidine	0.9155	1.29	0.8463	1.16	0.8064	1.16	0.6738	1.26
Arginine	6.9280	9.79	7.9908	10.99	7.7771	11.17	5.8317	10.87
Proline	3.4244	4.84	3.3691	4.63	2.8251	4.06	0.6499	1.21
Total amino acid content	70.7573	100	72.7068	100	69.6021	100	53.6613	100

Figure 7.

The comparison of different samples' amino acid content (1).

Figure 8.

The comparison of different samples' amino acid content (2).

From Table 1, no noticeable differences were found in the amino acid composition of the untreated wool sample and sample A, especially the content of cysteine. This showed that the lead acetate solution would not affect the amino acid contents of wool fibers, so it can be used to protect the thiols on wool in amino acid composition test. Sample B and sample D were also compared in Figure 7. After the LKS-610 reagent treatment, the fiber in sample B did not react with lead acetate and its cystine mass percentage was 3.80%. In sample D, however, the cystine content was reduced to 1.55% after the treated fibers reacted with lead ions. Test data demonstrated that part of the thiols would be oxidized to disulfide bonds again when wool fibers were separated from the reductive environment. This change will affect the accuracy of the LKS-610 reagent's reduction effect test. Therefore, before using amino acid composition test to verify the reducing effectiveness of LKS-610 reagent, the thiol groups should first be protected by lead ions.

The changes of the amino acid content of each fiber sample after treatment by LKS-610 reagent for different time periods are shown in Table 2 and Figure 8. After processing for 10 min by LKS-610 reagent, the cystine contents of wool fibers decreased significantly and its quality percentage reduced from 10% to 3.45%. With the increase of the processing time, the mass percentage of cystine decreased. When the processing time was 90 min, the sample's cystine percentage was only 1.03%. This change of cystine illustrated that LKS-610 reagent cleaves the disulfide bond effectively and causes the wool fiber's surface to become rich in thiols. Additionally, when processing time was longer than 60 min, the cystine content of samples did not change significantly.

The other amino acids contents did not decrease significantly in the test, it also means that the LKS-610 reagent does not affect other chemical bonds.³⁶ In summary, LKS-610 reagent has a significant effect on the disulfide bonds of wool fibers. It could break down most of disulfide bonds and generate thiol groups under the condition of 80℃ for 60 min. This treatment method would not excessively disrupt the structure of protein macromolecular chains and can be used for the preparation of a keratin solution with high molecular weight.

LKS-610-treated fibers dissolved in formic acid solution

The untreated and treated wool fibers were dissolved in the formic acid solution with the bath ratio of 1:20 at 60℃, respectively. The change of fibers in the dissolution process is shown in Figures 9 and 10.

Figure 9.

The dissolving state of untreated wool fibers in formic acid solution. The sample dissolved for (a) 1 h; (b) 2 h; (c) 3 h; and (d) 4 h.

Figure 10.

The dissolving state of pre-treated wool fibers in formic acid solution. The sample dissolved for (a) 1h; (b) 2h; (c) 3h; and (d) 4h.

Figure 9 shows the microscopical photographs of wool fibers dissolved in the formic acid solution. After dissolved for 2 hours, the main body of fibers did not change significantly and only parts of scales were open and detached. As the dissolution time was prolonged, the fiber gradually disintegrated and formed many cone-shaped fibrils, but it still wasn't dissolved in the formic acid solution. The dissolution process of LKS-610-treated fibers is shown in Figure 10, the fibers rapidly swelled and decomposed in the solution. When the dissolution time reached 4 hours, most of the fibers had been dissolved by the formic acid. This experimental result indicated that when the disulfide bonds in wool were altered by LKS-610, the interaction force between protein chains was obviously weakened. Therefore, the treated fibers could be more easily and effectively dissolved in reagents such as formic acid and prepared into keratin solution. After the LKS-610-treated fibers were dissolved at 60℃ for 5 h, the dissolution ratio reached 75% and the keratin extraction yield was 80–85% (ratio to the mass of dissolved protein).

The molecular weight of keratin in formic acid solution was tested by gel electrophoresis analysis and the result is presented in Figure 11. It could be found that keratin molecular weights were distributed to 45 kDa ∼ 60 kDa and 14.4 kDa ∼ 20 kDa. The decrease of protein molecule chain was caused by the fracture of disulfide bonds and acid degradation, but some keratins still hold a larger molecular weight. This phenomenon indicated that LKS-610 did not cause excessive damage to wool fibers, and the keratin solution prepared by this reagent had research value for recycled protein materials.

Figure 11.

SDS-PAGE gel electrophoretogram of keratin. (a) molecular weight standard; and (b) keratin extracted from formic acid solution.

Conclusion

The reaction effect and mechanism of a new reductant (LKS-610 reagent) on disulfide bonds in wool fibers is discussed in this article. LKS-610 reagent can quickly and efficiently cleave the disulfide bonds in wool fibers and alter these bonds into thiol groups. The SEM images showed that the fiber scales were gradually disrupted during processing. When treatment reached a certain extent, most of the scales were stripped, but the fiber's cortical layer was not damaged. The reaction mechanism of the LKS-610 reagent and wool was studied by the lead ions reaction and XPS test. The results demonstrated that the disulfide bonds in fibers could be disrupted by LKS-610 reagent and altered to thiol groups, but these thiols were easily oxidized to regenerate disulfide bonds again. In addition, the lead ions could react with these thiols and avoided the reconstruction of disulfide bonds. Amino acid composition test indicated that the mass percentage of cystine decreased from 10.09% to 1.05%, when wool fibers were processed by the LKS-610 reagent at 80℃ for 60 min. At the same time, LKS-610 reagent did not affect other chemical bonds in the wool fibers. In conclusion, LKS-610 reagent can effectively act on the disulfide bonds in wool under certain conditions, and it will not have obvious decomposition and destruction effects on protein macromolecules. The results of this study will be helpful for further research on wool keratin solutions which could be used to prepare regenerated protein materials.

Footnotes

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship and/or publication of this article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The authors would especially like to thank the National Key Technology Support Program (Number 2014BAE01B00) for financial support.

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