Choline-Based Amino Acid ILsâCollagen Interaction: Enunciating Its

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Choline Based Amino Acid ILs-Collagen Interaction: Enunciating its Role in Stabilization/destabilization Phenomena Aafiya Tarannum, Jonnalagadda Raghava Rao, and Nishter Nishad Fathima J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b10645 • Publication Date (Web): 14 Dec 2017 Downloaded from http://pubs.acs.org on December 14, 2017

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The Journal of Physical Chemistry

Choline

Based

Interaction:

Amino

Acid

Enunciating

its

ILs-Collagen Role

in

Stabilization/Destabilization Phenomena Aafiya Tarannum, J. Raghava Rao, N. Nishad Fathima* Chemical Laboratory, CSIR-Central Leather Research Institute, Adyar, Chennai-600020, India

ABSTRACT. Given the potential for productive interaction between choline based amino acid ionic liquid (CAAILs) and collagen, we investigated the role of four CAAILs, viz., choline serinate, threoninate, lysinate, phenylalaninate and the changes mediated by them in the structure of collagen at different hierarchical ordering of the same viz., at molecular and fibrillar level. The rheological, dielectric behaviour and the secondary structural changes signify the alteration in triple helical structure of collagen at higher concentration of CAAILs. A marginal swelling and slight decrease in thermal stability of RTT collagen fibres was sighted for choline serinate and threoninate, albeit distortions in banding patterns were noticed for choline lysinate and phenylalaninate suggesting chaotropicity of the ions at fibrillar level. This signifies the changes in the hydrogen bonding environment of collagen at the increasing concentration of CAAILs, which could be competitive hydrogen bonding between the carbonyl group of amino acid ILs and hydroxyl groups of collagen.

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1. INTRODUCTION Ionic liquids (ILs), regarded as “designer” or “task specific” solvents, can be tuned depending on the field of application1. Based on their outstanding properties and enormous combinations of cations and anions, ILs have emerged as refreshing class of compounds in electrochemistry, organic synthesis, catalysis, gas separation, protein preservation2-5 and so on. However, among a large number of ILs that had been synthesized and characterized, the conventional ILs like imidazolium have been demonstrated to be poorly biodegradable and have low biocompatibility, which can be a serious drawback in spite of their other excellent properties6-7. Amino acids, one of the ample biomolecules in nature, are nontoxic, biodegradable and biocompatible8. It has fetched substantial stance in the recent literature owing to their ubiquitous applications in modern chemistry, materials science and biosciences9-11. They are excellent materials for the synthesis of ILs, owing to their biodegradability and biological activities. The novel room temperature amino acid ILs (AAILs) was first synthesized by Fukomoto and co-workers by coupling the imidazolium cation with 20 different amino acids12. Designing, preparation and characterization of AAILs either as cations or anions was reported by Ohno and co-workers13. This in turn paved way for designing more biocompatible ILs for specific applications in biological, medical and pharmaceutical sciences14. Nevertheless, on the theoretical front, approaches based on density functional theory has been chiefly used in unraveling the interactions between amino acids and amino acids ester substituted derivatives ILs15 and the promising solvating properties for protein has also been proposed16. Structural stability of myoglobin was studied indicating a huge transformation to complete beta sheet from its native form using hydrated AAILs17. EMIM glycine, an effective alternate for imidazolium chlorides has been reported for the dissolution and reconstitution of silk18 whilst,


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the favorable stability of haemocyanins was observed in choline amino acids further enhancing its antiproliferative activity19. CAAILs have been considered as effective sorbents for CO2 capture due to their striking properties such as low toxicity, biodegradability and fast reactivity towards CO220. Yazdani et. al., reported the microbial biocompatibility and biodegradability of CAAILs in industrial sewage water, where EC50 values found in the range of 160-220 mg/L, which is regarded to be practically harmless based on hazard rankings21. They have been also reported as effective functional ingredients in topical formulations for drug delivery, by maintaining its stability and enhancing their solubility22. Current challenge is to functionalize CAAILs to have better performance. Collagen, a chief structural protein has its applications in numerous fields such as pharmaceutical, biomedical, food, cosmetics, leather etc. This further evinces interest in studying the interaction of collagen with varying ILs, such that the properties can be garnered and tuned for various applications ranging from biomedics to leather23-25.The interaction of collagen with ILs has been appraised with varying ionic liquids ranging from conventional cations such as imidazolium, phosphonium, ammonium to biocompatible cation choline along with varied anions. Imidazolium, phosphonium and ammonium ILs had destabilizing effect on collagen due to the chaotropicity of anions26-28, albeit choline dihydrogen phosphate stabilized collagen thus making it suitable for preparation of biomaterials29. Also, choline salts exhibited superior biocompatibility required for wound healing applications30. In addition to, imidazolium ILs demonstrated unhairing cum fibre opening of skin matrix, which has opened avenues for cleaner and greener existing processing systems31-32. The present work focuses on unraveling the underlying interactions choline based amino acid ILs (CAAILs) on the stability of collagen at different hierarchical ordering, ie at molecular and


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fibrillar level. The choline cation and amino acid based anions were selected as both are biodegradable in nature. Of the amino acids; serine, threonine, lysine and phenylalanine were selected, serine and threonine are polar, hydrophilic with hydroxyl group, lysine is polar, hydrophilic with basic group and phenylalanine is non-polar, hydrophobic with aromatic group. The intent of the study is to explore the interaction of different amino acid based ILs with collagen to understand their influence on stabilization and destabilization of collagen using various characterization techniques viz., FT-IR, CD, viscosity, thermal and dimensional stability and impedance. This would throw light on how to tune the anion and cation present in ILs to achieve the end property expected on collagen. 2. MATERIALS AND METHODS 2.1 Materials Rat tail tendons (RTT) were excised from albino rats (Wistar Strain), which was used for extraction of type I collagen. Choline threoninate (CT), choline serinate (CS), choline lysinate (CL) and choline phenylalaninate (CP) were procured from Ionic Liquid Technologies GmbH (IoLiTec, Germany). Millipore water was used for this study and all the chemicals used were of analytical grade. 2.2 Isolation of Type I collagen The extraction of type I collagen was carried out from tails of six month old albino rats (Wistar strain). 0.9% NaCl was used to wash the teased collagen fibers. The teased collagen fibers were dissolved in 0.5 M acetic acid at 4˚C with overnight stirring. It was precipitated using 5% sodium chloride, which was followed by centrifugation for garnering the precipitates. Then, the dialysis was carried extensively against 50 mM phosphate buffer and the collagen was re-dissolved in 0.5


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M acetic acid followed by final dialysis with 0.05 M acetic acid. Collagen concentration in solution was determined through hydroxyproline content by Woessner method33. 2.3 Preparation of collagen-CAAILs composites Collagen-CAAILs (CT- Choline Threoninate, CS- Choline Serinate, CL- Choline Lysinate and CP- Choline Phenylalaninate) of four different concentrations (1:0.05%(v/v)-1), (1:0.1%(v/v)-2), (1:0.5%(v/v)-3) and (1:1%(v/v)-4) were prepared in acetate buffer(pH 4) and continuously stirred for 3 hours at 4˚C followed by overnight incubation. The physico-chemical interaction patterns were studied for the incubated collagen-CAAILs composites. 2.4 CD studies CD spectra were recorded on a Jasco-815 Spectropolarimeter to observe the interaction for native collagen and collagen-CAAILs composites in the range 190-260 nm in a rectangular quartz cell with an optical path of 0.1 cm. 2.5 FT-IR studies FT-IR spectra were recorded on Jasco FT/IR-4200 infrared spectrometer. Lyophilized samples of native collagen and collagen-CAAILs composites were studied by KBr pellet method over the spectral range of 4000-400 cm-1, with a resolution of 4 cm-1 with an averaged 40 scans for each spectrum. 2.6 Viscosity measurements The rheology of native collagen and collagen-CAAILs composites was carried out using a Brookfield DV-II+Pro viscometer. 2.7 Impedance measurements Impedance analysis for native collagen and collagen-CAAILs composites were performed using CH Instrumental (USA) electrochemical analyzer CH-model 660 B to assess the effect of


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CAAILs on the resultant dipole of the collagen responding to an alternating electric field using the three classical electrode system, wherein, the glassy carbon, a platinum and saturated calomel electrode serves as a working electrode, counter electrode and as reference electrode respectively. Dielectric data can be depicted in terms of admittance Y″ (unit: Ω-1). It can be written as ∗ = + " where Y′ is the real component describing the energy stored and Y″ is the imaginary component depicting the energy dissipated by the system. 2.8 Thermal stability for RTT collagen fibers Denaturation temperature for native and CAAILs (CT-Choline threoninate, CS-Choline serinate, CL-Choline lysinate and CP-Choline phenylalaninate) treated RTT were studied using differential scanning calorimeter (Netzsch-DSC 204 F1 phoenix). Samples were blot-dried and were sealed in the hermetic aluminum Tzero pans and heated from 30-80˚C at the rate of 5˚C min-1. 2.9 Dimensional stability for RTT collagen fibers Dimensional stability for native RTT and RTT treated with CAAILs (CT-Choline threoninate, CS-Choline serinate, CL-Choline lysinate and CP-Choline phenylalaninate) at 25˚C for 24h was monitored under Aven Inc., Digital Mighty Scope, 1.3 M (Product code: 48708-25, Made in Taiwan) of 10x resolution. 3. RESULTS AND DISCUSSION 3.1 CD spectra ascribing changes in the secondary structure of collagen Changes in the secondary structure of collagen upon interaction with CAAILs were assessed using circular dichroic spectroscopic studies at molecular level. In the far UV region, native


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collagen exhibits its maxima at 222 nm and minima at 197 nm. The peak at 222 nm is a typical hallmark of type II Polyproline (PPII) conformation of collagen34. Figure 1 shows the CD spectra for native and CAAILs treated collagen. It was observed that there was a decrease in molar ellipticity values for CAAILs, when compared to native collagen. This might be owing to the stronger interaction of collagen with that of anions viz., serinate (C-CS4), threoninate (C-CT4) and lysinate (C-CL4). For phenylalaninate (C-CP4), there was a complete red shift in peak at 197 nm, which also implies the changes in the hydrogen bonding environment causing structural deformity in collagen, whereas for C-CS, C-CL and C-CT (4) doesn’t show any distinctive changes. It is said that the collagen on complete denaturation evidences the disappearance of positive peak at 220 nm and the negative band shifts towards higher wavelengths. This outcome entails that the anions are the reporters of structure in ionic liquids, whereas the cations play minimal role as also reported earlier in literature28, 35. The CD spectrum for native collagen, CCS (1-3), C-CT (1-3), C-CL (1-3) and C-CP (1-3) is given in supplementary section (S1). The parameter Rpn, a characteristic ratio for triple helical conformation of collagen denotes the ratio of positive peak intensity over negative peak intensity. The Rpn ratios for collagen and CAAILs treated collagen are given in table 1. From the table, it is evident that there was a continuous increase in Rpn values for C-CP (1-4) with the increase in concentration. For C-CL, C-CT and C-CS (1-3) there was no significant increase or decrease in Rpn values compared to native collagen, whereas for higher percentages C-CS, C-CL, C-CT (4), there witnessed a slight increase in Rpn values with that of native collagen. Rpn values for C-CS, C-CP, C-CL and C-CT (1-3) are given in supplementary section (Table 1). It could be ascertained that the increase in CP concentration resulted in major changes in the CD spectra, witnessing the red shift for both positive and negative peaks. This also indicates that the changes in the CD spectra of collagen in


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the presence of CAAILs might be due to the loss of triple helicity of collagen, as it is well known that collagen on partial or complete degradation undergoes drastic changes like the shifting and disappearance of positive and negative peaks.

Figure 1. CD spectra for native collagen and collagen treated with C-CS4, Collagen: Choline Serinate (1:1%), C-CT4, Collagen: Choline Threoninate (1:1%), C-CP4, Collagen: Choline Phenylalaninate (1:1%), C-CL4, Collagen: Choline Lysinate (1:1%), molar ellipticity at 222 nm (inset) Table 1. Rpn ratio for native collagen and collagen treated CAAILs Collagen: CAAILs

Rpn (characteristic ratio)

Native collagen

0.122

C-CS4

0.137

C-CT4

0.139

C-CP4

0.487

C-CL4

0.132


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3.2 FT-IR spectra ascribing changes in the functional groups of collagen Table 2. FT-IR spectra for native collagen and collagen treated with C-CS4, Collagen: Choline Serinate (1:1%), C-CT4, Collagen: Choline Threoninate (1:1%), C-CP4, Collagen: Choline Phenylalaninate (1:1%), C-CL4, Collagen: Choline Lysinate (1:1%) BANDS

Amide A

Amide B

Amide I

Amide II

Amide III

Native

3300

3173

1640

1560

1240

CS-(1:1%)

3285

3173

1643

1552

1237

CT-(1:1%)

3285

3172

1640

1555

1239

CL-(1:1%)

3280

3170

1635

1554

1239

CP-(1:1%)

3280

3170

1635

1549

1240

Table 2 explicates the FT-IR bands observed for CAAILs (1:1). Collagen shows the distinctive peaks at 3300 cm-1 for amide A, 3170 cm-1 for amide B, 1640 cm-1 for amide I, 1560 cm-1 for amide II and 1240 cm-1 for amide III36. Distinctive peaks for serinate, threoninate, lysinate and phenylalaninate was observed at 3200 cm-1, which corresponds to NH stretching, peaks around 3100 and 3029 cm-1 reads for OH stretching indicating hydroxyl groups, peak at 1690 and 1020 cm-1 accounts for carboxylic acids, peak around 1660 cm-1 corresponds to C=O stretching, peak at 3020 cm-1 for aromatic C-H stretching and 1575 cm-1 accounts for aromatic C-C bending. N-H stretching vibrations for collagen treated CAAILs denotes amide A and amide B with a frequency of 3286 cm-1 and 3100 cm-1, respectively. They are centered on NH groups and they depend on the strength of hydrogen bonding. It discloses the negligible shifts in peaks for amide A from 3286 cm-1 to 3280 cm-1. Frequency of amide B was found to be decreased, which suggests a feasible interaction for all CAAILs with NH groups of the collagen. The C=O


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stretching vibrations is apparent for amide I, which counts on the backbone structure of collagen. A band ranging around 1635-1643 cm-1 affirms shifts at 1635 cm-1 for the CP and CL compared to that of control (1640 cm-1). Amide II band renders information on C-N stretching vibration. Native collagen exhibits a frequency around 1560 cm-1, whereas a slight shift in bands to 1549 cm-1 was observed at the increased concentration of CAAILs indicating the stronger interaction with that of collagen. Amide III band representing NH bending and CN stretching vibrations was identified at 1240 cm-1. There was negligible shift in peaks for amide III and this could possibly be owing to microenvironmental changes in collagen. FT-IR spectra for C-CS, CT, CL and CP (1-4) are given in supplementary figure (S2). The peak around 3200, 3100, 1690, 1660, 3020 and 1575 cm-1 signalizes the presence of NH groups, hydroxyl groups, carboxyl groups, aromatic groups respectively, which is distinctive for amino acids. From the experimental observations, it was concluded that the ions interact strongly with functional groups of collagen resulting in structural deformity, which was also observed from CD studies. 3.3 Viscosity measurements ascribing changes in the flow rate of collagen In order to understand the interaction between collagen-CAAILs composites absolute viscosity for native and CAAILs treated collagen was carried out. The absolute viscosity for native collagen was found to be around 1.8 cP (figure 2). A slight increase in viscosity with the increase in concentration of collagen-CAAILs (0.05%-1%) was witnessed indicating aggregation. Increase in viscosity of the protein generally attributes to aggregation of protein with additives37. Hence, the CAAILs interaction with collagen resulted in increased viscosity. This might be due to the competitive hydrogen bonding between the functional groups of collagen and CAAILs thereby restricting the flow. It can also be due to the exposure of hydrophobic residues leading to collagen aggregation.


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Figure 2. Viscosity for native collagen and collagen treated with C-CS, Collagen: Choline Serinate (1:0.05% to 1:1%), C-CT, Collagen: Choline Threoninate (1:0.05% to 1:1%), C-CL, Collagen: Choline Lysinate (1:0.05% to 1:1%), C-CP, Collagen: Choline Phenylalaninate (1:0.05% to 1:1%) 3.4 Impedance measurements ascribing changes in the hydration behavior of collagen The dielectric properties and hydration behavior of biomacromolecules with varying concentrations of CAAILs were inferred from impedance measurements. This technique is exceptionally sensitive to appraise the dynamics of water molecule. Since, the favorite environment for biomolecules is water, which is said to be powerful H-bonding medium and has strong interactions with water. It is well known that the hydrophilic groups of protein molecules resides on the surface, which have a strong affinity to interact with water molecules from its surrounding milieu whereas hydrophobic groups are buried in the interior core of protein moieties. The change in dielectric dipole or dipole moment cogitate the mobility of functional groups, which in turn predicts the structural stability of protein. The dipole moment of a protein molecule is owing to the positive and negative charges, which results from the presence of acidic/basic amino acid side chains. This usually depends on the pH of the solution; intra-


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molecular ability and molecular conformation as each polar entity renders distinctive response towards an applied electric field38. The change in the dielectric behavior of collagen on interaction with CAAILs has been plotted (Figure 3). Figure 3 shows the Nyquist admittance plot, which demonstrates the ability to trap electric charge through the steepness of circles. Nyquist admittance plot was plotted as real Y’ vs. imaginary Y”39. Collagen has shown the lowest permittivity, whereas the concentration dependent permittivity was observed for all CAAILs. Decrease in the permittivity with the increase in concentration of CAAILs indicates the alteration in the polarizability of functional groups, which in turn destroys the dipolar nature of protein molecules. This might be owing to the fact that collagen is a protein with functional groups, leading to its charged behavior. Collagen carries a net positive charge on its triple helical structure at pH 4.5; the hydration shell of the protein molecule is tuned with the CAAILs concentration after complexation with CAAILs. It is likely that CAAILs may be involved in hydrogen bonding with the side chains of collagen. The bond formation alters the water structure around the functional group, which leads to the reorientation of protein moieties. CAAILs alter the hydrophilic interaction between the triple helix, signifying the rearrangement of structural water around the collagen. It could be due to the strong effect of CAAILs, which leads to the reorganization of bound water molecules in the collagen. It also explicates the bound charges in the protein when comes in contact with dielectric current brings about the reorientation and reorganization in the network of proteins moieties.


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B

A

C

D

Figure 3. Impedance measurements for native collagen and collagen treated with (A) C-CS, Collagen: Choline Serinate (1:0.05% to 1:1%), (B) C-CT, Collagen: Choline Threoninate (1:0.05% to 1:1%), (C) C-CP, Collagen: Choline Phenylalaninate (1:0.05% to 1:1%), (D) C-CL, Collagen: Choline Lysinate (1:0.05% to 1:1%) 3.5 DSC measurements ascribing changes in the thermal behavior of RTT collagen fiber Rat tail tendons (RTT) said to be the rich source for type I collagen were teased from albino rats. At fibrillar level, thermograms for RTT treated with CS, CT, CL and CP (1%) has been assessed using differential scanning calorimetry (Figure 4). Denaturation temperature (Td) for native RTT collagen fiber is 63˚C as cited in the literature40. The witnessing decrease in thermal stability for CS, CT, CP and CL (1%) on RTT collagen fibers was observed compared to native


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RTT, indicating the stronger interaction of anions with that of RTT collagen fibers. The decrease in thermal stability validates the changes in the hydration network of RTT collagen fibers. Any kind of alteration in the surrounding milieu of collagen with CAAILs brings changes in the structure of collagen fibres, viz., reorientation in the H-bonding network leading to the destabilization of collagen fibres. DSC thermograms for RTT treated with choline serinate, threoninate, phenylalaninate and lysinate (0.05-0.5%) is shown in supplementary figure (S3).

Figure 4. DSC thermograms for native RTT and RTT treated with Choline Serinate, CS-4-(1%), Choline Threoninate, CT-4-(1%), Choline Phenylalaninate, CP-4-(1%), Choline Lysinate, CL-4(1%) 3.6 Dimensional micrographs ascribing changes in the dimensions of RTT collagen fibers RTT type I collagen fibrils exhibits typical banding pattern unveiling the helicity of fibrils using dimensional microscopy41. Figure 5 depicts RTT treated with CS, CT and CP-1% revealing the marginal swelling within the incubated hour. However, there were no changes observed in the later hours. For CL-1%, huge swelling in RTT collagen fibers was noticed indicating the chaotropicity of anions leading to the distorted wave pattern resulting in agglomeration of RTT collagen fibers, which was also confirmed from the decreased thermal


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stability. RTT treated with CS, CT, CL and CP (0.05, 0.1 and 0.5%) is shown in supplementary figure (S4).

Figure 5. Dimensional micrographs for native RTT and RTT treated with Choline Serinate, CS(1%), Choline Threoninate, CT-(1%), Choline Phenylalaninate, CP-(1%), Choline Lysinate, CL(1%) 4. Conclusions In summary, this study elucidates the interaction between collagen and CAAILs at different hierarchical orderings viz., at molecular and fibrillar level. It was demonstrated that conformational changes in the helices of collagen was observed indicating destabilization at higher levels. It was worth noting that upon interaction of collagen in CAAILs, there was a noticing decrease in the molar ellipticity values for the 1% CAAILs with the complementing FTIR spectra, where witnessing shifts in peaks was observed for 1% CAAILs. Dielectric properties of collagen treated with CAAILs sheds light on the solvating behavior, indicating the reorganization and reorientation of protein moieties in CAAILs. The decrease in thermal stability and increased swelling of RTT collagen fibers with the increasing concentration of CAAILs


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equilibrates with the molecular level studies indicating the structural deformity of collagen. It is hypothesized that CAAILs and collagen both contains C=O moiety, N-H groups and O-H groups as side groups. Collagen may form H-bonds with CAAILs viz., between the carbonyl group of amino acids and hydroxyl groups of collagen therefore leading to the structural deformation of collagen. Supporting Information Available: This study proves the interaction of choline based amino acid ionic liquids with collagen at different hierarchical orderings through experimental studies. Author information Corresponding Author Tel.: +91 44 24437188, E-mail address: [email protected], [email protected] (N.N. Fathima) Acknowledgment We thank CSIR-RIWT (Research Initiatives for Waterless Tanning), a 12th five year plan project for providing the fellowship. CLRI Communication Code: 1239. Abbreviations CAAILs, Choline based amino acid ionic liquids; RTT, rat tail tendons; CD, circular dichroism; FT-IR, fourier transform infrared spectroscopy.


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