Preparation of Keratin-Glycine Metal Complexes and Their Scavenging Activity for Superoxide Anion Radicals

To address the problem of limited application of natural SOD, the development of SOD mimic enzymes is of great importance for bioantioxidation. Herein, we report on a new type of biopolymer antioxidant with excellent scavenging activity for O2 , keratinglycine metal complexes (FK-GlyM, M = Zn, Cu, Mn, Ni). They are prepared by feather keratin firstly combined with glycine and then metal ions. Using FT-IR, TG, CD, and SEM, the performance of the obtained complexes (FK-GlyM) for scavenging O2 ∙− is analysed and investigated. Importantly, the scavenging activity of FK-GlyCu is excellent in all FK-GlyM, and FK-GlyCu60 has the most excellent anti-O2 ∙− activity in all FK-GlyCux, of which EC50 and degree of simulation were, respectively, up to 4.5 × 10 −3 ± 0.0012 μmol/L and 911.1% compared with nature Cu, Zn-SOD. Finally, its mechanism was also discussed. In summary, this method about the simulation strategies will provide a novel idea for exploiting new-type biocompatible and highly reactive antioxidants.


Introduction
In vivo, excess oxygen free radical is harmful to the body [1].Under normal circumstances, the production and the elimination of oxygen radicals are balanced [2].However, due to the impact of the disease, age, and the external environment, excess oxygen radicals would be accumulated in the cells when the physiological balance was broken [3,4], leading to host tissue be damaged [5], such as the damage of protein, lipid peroxide, DNA mutation, and enzyme inactivation.However, superoxide dismutase (SOD) as antioxidant is important to protect cells from oxidative damage [6], which can catalyze the disproportion action of superoxide anion radicals (O 2 •− ) and further balance O 2 •− in vivo.Although the natural SOD has the excellent activity for scavenging O 2 •− , many disadvantages still exist [7], such as complex extract process, high cost, big molecular weight, easy deactivation, and not easily entering the cell membrane, which limited its application in medicine and other fields [8][9][10].Therefore, the preparation of SOD mimic enzyme is becoming more and more popular [11,12].
Currently, a lot of enzyme simulations for scavenging O 2 •− have been synthesized, including porphyrin metal complexes [7,13], macrocyclic metal complexes [14], and Schiffbase metal complexes [15].However, due to the low solubility in water, large side effects, and so on, the widespread applications of these SOD mimics based on micromolecule were still limited.Therefore, SOD mimics based on macromolecule become the new direction for simulation of SOD.In SOD mimic based on polymer, there are mainly cyclodextrins [16] and proteins [17][18][19].For proteins, they can form directly complexes with metal ions since they contain a large number of amino and carboxyl functional groups with complex function.In general, those metals as SOD activity center mainly include Cu [20], Co [21], Mn [22,23], and Fe [24].Ibrahim et al. [25] reported that Ovotransferrin (OTF) had function of scavenging O 2 •− when it combined with Cu and Mn.Besides, there is an important complement oxygen free radical scavenger in vivo: Metallothionein (MT).It is a class of protein containing low molecular weight and abundant thiol groups, which contribute to scavenging O 2 •− by combining with metal ions [26].

International Journal of Polymer Science
Keratin widely exists in hair, feathers, wool, nails, and horns [27].Due to its special molecular structure, it is hard to be used by humans and casually abandoned, which caused the waste of resources and bad environmental pollution [28].However, they have a large number of disulfide bonds [29,30].If thiol groups produced by the reduction of disulfide bonds combine with metal, the structural system similar to metallothionein will be generated.In this paper, feather keratin (FK) and glycine (Gly) firstly were combined to generate FK-Gly in an aqueous solution, then the transition metal ions (Zn II , Cu II , Mn II , and N II ) were combined with FK-Gly to generate keratin-based biopolymer metal complexes (FK-GlyM, M = Zn, Cu, Mn, Ni) and further studied its property of scavenging O 2 •− and seriously explored its mechanism.

Equipment for Characterization.
Fourier transform infrared spectra (FT-IR) were measured by an FT-IR spectrometer (FTS 3000) of DigiLAB, and the transmittance spectra were collected in the range of 4000-400 cm −1 at a resolution of 0.008 cm −1 .Thermogravimetric analyses of products were carried out on a PerkinElmer Pyris Diamond thermogravimetry (TG)/dynamic thermal analyzer at a heating rate of 10 ∘ C/min under a nitrogen atmosphere.Circular Dichroism (CD) was recorded by a Jasco J-810 spectropolarimeter (1 cm cell).The wavelength of scanning was from 200 to 250 nm, the speed was 200 nm/min, and a response time was of 1 s.The morphologies of product were studied by scanning electron microscopy (SEM) (Zeiss ULTRA Plus microscope, Germany).

Preparation of Keratin-Glycine Complexes (FK-Gly).
Firstly, certain keratin, Na 2 S 2 O 3 , and glycine were dispersed into water, and then certain concentration of NaOH solution was added to adjust the pH up to 7-8.Finally this mixture was stirred 6-12 h in the dark at room temperature.The keratinglycine complex (FK-Gly) was successfully prepared.

Preparation of Keratin-Glycine Metal Complexes (FK-GlyM x ).
The preparation process of FK-GlyZn 5 is as follows: Zn (OAc) 2 and FK-Gly were added to simple tube (M FK-Gly : M Zn = 1 : 5).After being magnetic stirred 12 h in the dark, FK-GlyZn 5 was obtained and set aside in the dark at 0-4 ∘ C. The concentration of FK-GlyZn 5 was calculated according to the amount of FK.The preparations of FK-GlyCu 5 , FK-GlyMn 5 and FK-GlyNi 5 are similar to that of FK-GlyZn 5 .

Results and Discussion
The preparation route of FK-GlyM was shown in Scheme 1, where the FK and FK-SH were the partially crosslinking protein and reduced protein.FK-Gly was modified by Gly and FK-GlyM was keratin-glycine metal complex.Firstly, the disulfide bonds (-S-S-) in keratin firstly were reduced to thiol groups (-SH) by strong reductant, and then these thiol groups reacted with the amino of glycine to generate keratin-glycine complex.Finally, keratin-glycine complex reacted with metal to give keratin-glycine metal complex.

Structure Characterization of FK-GlyM.
The obtained FK-GlyM was characterized by FT-IR spectra, TG, CD, and SEM.Moreover, we also discussed the mechanism of antioxidation activity.

FT-IR Spectra.
The prepared keratin-glycine Cu complex (FK-GlyCu 60 ) was frozen in the refrigerator at −20 ∘ C for 24 h.After being lyophilized, the products and KBr were mixed with 1 : 100 and made into transparent tablet for measuring FT-IR spectra.The result was shown in Figure 1.In FT-IR spectrum of FK, some characteristic absorption peaks at 1647 cm −1 , 1522 cm −1 , and 1236 cm −1 were, respectively, assigned to amide I, II, III band of FK, which indicated that the secondary structure of FK was mainly free coil.However, for FK-GlyCu 60 , the characteristic absorption peaks of amide I, II, III band demonstrated that the secondary structure of FK-GlyCu 60 was mainly free coil and -sheet structure.Therefore, the secondary structure of FK had been changed, and some random coil structures also had been into -sheet structure in the preparation process of FK-GlyM.Besides, characteristic absorption peaks of S-S bonds had happened red shift from 580 cm −1 to 546 cm −1 , which illustrated that S-S bonds had become less stable in the preparation process.It could be conjectured that some S-S bond had shifted and might generate -SH or sulfur anion in preparation process.

TG Analysis. TG curves of FK and FK-GlyCu
Scheme 1: The preparation process of FK-GlyM (M = Zn II , Cu II , Mn II , Ni II ).stability of the obtained keratin-glycine metal complexes was reduced.

CD Analysis.
Circular Dichroism (CD) of FK-GlyCu  (0.005 mM) was measured at the wavelength of 190-300 nm and room temperature.The results were shown in Figure 3.The characteristic peaks of random coil structure of FK appeared at near 195 nm while the characteristic peaks of -sheet layer structure of FK-GlyCu  complex appeared in the vicinity of 200 nm.Therefore, it was showed that the secondary structures of the prepared FK-GlyCu  were mainly -sheet and random coil, which could be attributed to the fact that the part random coil of secondary structure of the FK-GlyCu  complex had transformed into -sheet compared with pure FK.The result was the same as FT-IR spectrum analysis.Cond: [FK] = 5 mol/L, n (FK) : n (Gly) = 1 : 0.2;  = 20 ∘ C.

SEM Images.
After being lyophilized, the microstructure of dry FK and FK-GlyCu 60 was characterized by scan electron microscopy (SEM).As showed in Figure 4, in contrast to FK, it was found that they had the porous support structure and the pore size was 0.2-2 m in FK-GlyCu 60 .These porous structures would facilitate the adsorption of small molecules and macromolecular compounds.

O 2
•− Scavenging Activity of FK-GlyM.Using NBT photochemical reduction method to investigate scavenging superoxide anion radicals (O 2 •− ) activity of keratin (FK) and the keratin-glycine complex (FK-Gly), the results were shown in Table 1 and Figure 5.According to the results, FK and FK-Gly ([FK] = 5 mol/L) both presented some anti-O 2 •− activity, and the scavenging activity of FK-Gly was better than FK.Compared with FK, the activity of FK-Gly was doubled.

O 2
•− Scavenging Activity of FK-Gly under Different Environment.In order to get better FK-Gly, the effect factors on its scavenging activity, such as temperature, pH, and Gly content, were investigated.
At Different Temperature.Under the condition of the same ratio, the effects of temperature on the scavenging activity of FK-Gly for O 2 •− were investigated.The results were shown in Table 2 and Figure      At Different pH Value.Under the condition of the same ratio and temperature, the effects of different pH on the scavenging activity of FK-Gly for O 2 •− were investigated.The results were shown in Table 3 and Figure 7, which indicated that the scavenging activity of FK-Gly was best when the pH was 7-8 and EC 50 and degree of simulation could, respectively, up to 0.23 ± 0.03 mol/L and 17.8%.and the results were shown in Table 4 and Figure 8.It was found that the scavenging activity of FK-Gly was best and EC 50 and degree of simulation could, respectively, reach 0.21 ± 0.06 mol/L and 19.5% when the ratio of FK and Gly was 1 : 0.6 though the scavenging activity was similar when the ratios of FK and Gly were 1 : 0.2-1 : 0.6.

O 2
•− Scavenging Activity of FK-GlyM.Similarly, adopting NBT photochemical reduction method investigated anti-O 2 •− activity of FK-GlyM 5 (M = Zn, Cu, Mn, Ni).Although International Journal of Polymer Science they all had certain anti-O 2 •− activity, the different metal complexes had different activity.To compare anti-O 2 •− activity and EC 50 of different FK-GlyM in Table 5 and Figure 9, the results revealed that FK-GlyCu 5 had the most active for scavenging superoxide anion radicals, of which EC 50 was 0.067 ± 0.0014 mol/L and degree of simulation was 61.2% comparing with nature Cu, Zn-SOD.However, the activity of FK-GlyZn 5 was most weak and its EC 50 was only 0.24 ± 0.08 mol/L and degree of simulation was 17.1% comparing with nature Cu, Zn-SOD.To sum up, the scavenging activity for O 2 •− of four kinds of keratin-glycine metal complexes was FK-GlyCu 5 > FK-GlyMn 5 > FK-GlyNi 5 > FK-GlyZn 5 .
The results indicated that the mount of metal ions (Cu II ) had important effect for anti-O 2 •− activity of FK-GlyCu  complex.With the increase of metal ion Cu II , the anti-O 2 •− activity of FK-GlyCu  was first increased and then decreased.
The scavenging activity of FK-GlyCu 60 for O 2 •− was strongest in Table 6 and Figure 10.Compared with the literature [31], of which EC 50 and degree of simulation, respectively, were 0.038 ± 0.003 mol/L and 107%; here EC 50 of prepared FK-GlyCu 60 was far below it and the degree of simulation was much bigger than it.The values, respectively, were up to 0.0045 ± 0.0012 mol/L and 911.1%.

Suggested Mechanism of Active
Center.According to our reported results, the active center and mechanism of FK-GlyM for scavenging O 2 •− were suggested.There exist possibly three active reaction centers (Scheme 2).( 1) Metal ion and N from the histidine and amino acid chain of FK residues formed coordination bonds, obtaining a six-member ring structure which provided O 2 •− reaction centers.(2) Metal ions and N, O in amino acid chain of FK coordinated, respectively, to form a plurality of annular reaction center, and the synergy of multiple active centers improved the scavenging activity of FK-GlyM for O 2 •− .Meanwhile, due to the introduction of glycine, the aggregation of polymer chains in keratin became difficult and further increased the solubility of FK-GlyM.(3) For FK-GlyM, the partial COO − of glycine and M 2+ occurred to electrostatic attraction that led to form new reactive center.Moreover, the thiol (-SH) from S-S had active hydrogen atom which could cause the free radical of chain growth to become less active and further generate the free radical with poor reactivity or extremely stable inhibitor free radical via providing active hydrogen.In either of these two cases, the inhibitor slowed down the original radical reaction and even stopped the reaction carrying out.

Conclusion
In summary, a simple biopolymer antioxidant (FK-GlyM) with excellent scavenging activity has been gained via simply combining FK with Gly and metal ions.It was found that the scavenging activity of FK-GlyM for O 2 •− was prior to FK-Gly and FK.After exploring the effect of temperature, pH, and ratios on scavenging activity, the results proved that the optimal conditions were 20 ∘ C, pH = 7-8, and n (FK) : n (FK-Gly) = 1 : 0.6, where the scavenging capacity of FK-Gly for O 2 •− was best.In four kinds of FK-GlyM, it turned out that the order of scavenging activity was FK-GlyCu 5 > FK-GlyMn 5 > FK-GlyNi 5 > FK-GlyZn 5 .And FK-GlyCu 60 was proved to have the most outstanding anti-O 2 •− activity in all FK-GlyCu  , of which EC 50 and degree of simulation separately reached 4.5 × 10 −3 ± 0.0012 mol/L and 911.1% compared with nature Cu, Zn-SOD.The reutilization of discarded feather keratin to prepare biopolymer antioxidant points out a new idea for both the problem of resources environment and the research on biopolymer antioxidant to us.
60 were presented in Figure2.It could be seen that FK gave three weight losses within total temperature range, which could be assigned to the weight loss of adsorbed water at about 100 ∘ C and keratin decomposition at 200 ∘ C and 375 ∘ C.Meanwhile, FK-GlyCu 60 also gave three weight losses within total temperature range, caused by the decomposition of FK at 200 and 375 ∘ C as well as no weight loss after 575 ∘ C. In summary, compared with feather keratin, the thermodynamic

Figure 7 :Figure 8 :
Figure 7: The comparison of O 2 •− scavenging activity of FK-Gly at different pH values.

Table 2 :
EC 50 of FK-Gly at different temperatures.

Table 3 :
EC 50 of FK-Gly at different pH values.

Table 4 :
EC 50 of FK-Gly at different mounts of Gly.

Table 6 :
The effect of the mount of Cu II for O 2 •− scavenging activity of FK-GlyCu  .