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Stability of collagen in ionic liquids: Ion specific Hofmeister series effect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 212 (2019) 343–348
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Stability of collagen in ionic liquids: Ion specific Hofmeister series effect Aafiya Tarannum, Raghava Rao Jonnalagadda, Nishad Fathima Nishter ⁎ Inorganic and Physical Chemistry Laboratory, CSIR-Central Leather Research Institute, Adyar, Chennai-20, India
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Article history: Received 24 October 2018 Received in revised form 4 January 2019 Accepted 15 January 2019 Available online 16 January 2019 Keywords: Imidazolium-based ionic liquid Destabilization RTT collagen fibres Circular dichroism studies Collagen
a b s t r a c t In protein-ionic liquids (ILs) interactions, anions play an important role. In this work, imidazolium-based ILs (IILs) with varying anions namely dicyanamide (DCA), hydrogen sulfate (HS), dimethyl phosphate (DP), acetate (A), sulfate (S) and dihydrogen phosphate (DHP) have been chosen with the aim of understanding the role of anions in bringing about the destabilization effect on collagen based on the kosmotropicity and chaotropicity of ions. Imidazolium-based ILs destabilized the triple helical structure of collagen, thereby proving as strong denaturants for collagen and this was confirmed by various spectroscopic techniques viz., CD, FT-IR, viscosity and impedance measurements. The solution studies were in accordance to the changes in the dimensional stability of RTT collagen fibres at the fibrillar level. Imidazolium cations with varied anions have exhibited destabilizing effect on collagen in order of ions in Hofmeister series; IDP b IDHP b IA b IDCA b IS b IHS. Presumably, these notable effect and changes were facilitated by electrostatic interactions between the anions and amine functional groups of collagen. © 2019 Elsevier B.V. All rights reserved.
1. Introduction Ionic liquids, lime lighted as “green” and “designer” materials have instigated a paradigm shift in every field due to its unique and attractive physicochemical properties [1–2]. Depending on the cations and anions, they can be specifically and effectively tuned for a wide range of applications in green chemistry to pharmaceutical and biotechnological applications [3–4]. In general, a pair of strong kosmotropic anions and chaotropic cations stabilize the protein, whereas chaotropic anions and kosmotropic anions destabilize the protein and selection of ions are based on Hofmeister series [5]. Research on the interaction and behavior between ionic liquid and protein has nested stance in scientific community due to its remarkable properties. The spectral and thermodynamic behavior of IL-BSA system suggested the loss of secondary and tertiary structure and the anionic moiety seemed to have more obvious effect than the cationic ones [6] and the denaturation was caused by electrostatic and hydrophobic interaction, hydrogen bonding and van der Waals forces as proved by Zhu and Yan et al. [7–8]. The stability of insulin and heme protein in imidazolium based ILs with varying anions was also evaluated and it was disappointing to note that ILs failed to protect the native state, which led to denaturation [9–10]. Therefore, protein stability is receiving rapid interest in the active research in biological and physical sciences providing an adequate and fundamental understanding of the behavior and stability of proteins in ILs.
⁎ Corresponding author. E-mail address: [email protected] (N.F. Nishter).
https://doi.org/10.1016/j.saa.2019.01.029 1386-1425/© 2019 Elsevier B.V. All rights reserved.
Collagen, the most abundant protein in mammals has been used widely as a biomaterial [11–12] and they have been explored with ionic liquids [13–14]. The dissolution and regeneration of collagen using BMIM Cl studied by Meng et al. reported partial destruction in triple helical structure [15]. Zhang et al., has prepared cellulose/collagen film using [EMIM] [Ac] as common solvent [16] and they have been used in regenerating triple helical structure, which is indicative of potential applications in tissue engineering and it has been proved using experimental and computational studies [17–18]. We have studied the influence of various ionic liquids on collagen on the different hierarchical orderings of collagen. The influence of BMIM Cl has brought significant changes at higher hierarchical level of collagen [19]. At molecular and fibrillar level, choline amino acid based ILs has also demonstrated destabilizing effect due to competitive hydrogen bonding between its molecules [20]. Also, ammonium and phosphonium based ILs had demonstrated destabilizing effect on collagen due to their chaotropicity of ions [21–22], whilst choline dihydrogen phosphate stabilized collagen by exerting an electrostatic force, thus making it suitable for preparation of biomaterials for tissue engineering applications [23–24]. Also, the changes in the structure and hydration dynamics of collagen has been studied upon interaction with imidazolium and choline based ILs [25]. In addition to, imidazolium based ILs has been employed as unhairing cum fibre agent for skin matrix and it has paved way for cleaner and greener processing systems [26–27]. Still the interactions between proteins and ionic liquids in aqueous media are not well understood and the underlying interpretations are mostly speculative. This emphasizes the need to understand the interaction of collagen with various additives, thus providing a new probe for elucidating protein- IL interaction. Herein, a series of imidazolium-
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based ionic liquid (IILs) with wide range of varying anions such as dicyanamide, hydrogen sulfate, dimethyl phosphate, acetate, sulfate and dihydrogen phosphate has been comprehensively investigated at both molecular and fibrillar level. The choice of cations and anions was based on chaotropic and kosmotropic behavior of ions as described in Hofmeister series. The interaction of IILs with collagen has been studied using various characterization techniques viz., circular dichroic studies, Fourier transform infrared spectroscopy, viscosity and UV–Vis studies, dimensional stability and impedance measurements to influence the varying anions on collagen. 2. Experimental
the far-UV region ranging from 260 to 190 nm and it was scanned with 0.2 nm intervals with a path length 1 mm at 25 °C with computer averaged three scans for each sample. The data were obtained in milli degrees and further they are converted to molar ellipticity (deg·cm2·dmol−1). Molar ellipticity was plotted against wavelength in nanometers (nm). 2.7. FT-IR Studies Lyophilized samples of native and IILs treated collagen were studied using Jasco FT-IR-4200 (Fourier Transform Infrared Spectrometer) with 40 scans in the range of 4000–400 cm−1 and the resolution of 4 cm−1. The studies were carried out using KBr pellet method.
2.1. Materials 2.8. Impedance Measurements 1-Butyl-3-methylimidazolium dicyanamide (IDCA), 1-Butyl-3methylimidazolium hydrogen sulfate (IHS), 1-Butyl-3methylimidazolium dimethyl phosphate (IDP), 1-Butyl-3methylimidazolium acetate (IA), 1-Butyl-3-methylimidazolium sulfate (IS) and 1-Butyl-3-methylimidazolium dihydrogen phosphate (IDHP) were procured from Sigma Aldrich and Ionic Liquid Technologies GmbH (IoLiTec, Germany). Type I collagen was extracted from sixth month old albino rats. 2.2. Extraction of Type I Collagen Rat tail tendons were used for extracting the type I collagen. RTT were teased and washed thoroughly with 0.9% saline at 4 °C. Purification was carried out using 5% sodium chloride and centrifuged for garnering the precipitates. The precipitate obtained was redissolved in 0.5 M acetic acid and dialyzed against 50 mM phosphate buffer with pH of 6.5 ± 0.2. Then the precipitated collagen was re-dissolved in 0.5 M acetic acid with the final dialysis against 0.05 M acetic acid. Hydroxyproline content was done to estimate the concentration of collagen using method of Woessner [28]. SDS-PAGE was carried out to confirm the purity of collagen (Supplementary Fig. S1). The obtained collagen solution was stored at 4 °C. 2.3. Preparation of Collagen IILs Solution The collagen solution in acetate buffer was treated with varying concentrations of IILs. IDCA, IA, IDP, IHS, IS, IDHP [1–4] was stirred continuously at 4 °C for 3 h. The concentration are as follows; C-I1–1: 0.05%, C\\I2\\ (1:0.5%), C\\I3\\ (1: 5%), C\\I4\\ (1:10%) throughout to prevent denaturation of collagen due to heat generated during mixing. The working concentration of collagen was taken as 0.8 μM at pH 4.0. The concentration of collagen was kept constant and IILs concentration was varied. 2.4. Viscosity Measurements Viscosity measurements were carried out to study the influence of native and IILs treated collagen on the rheological property using a Brookfield DV-II + Pro viscometer at 25 °C. 2.5. UV–Vis Studies Native and IILs treated collagen was appraised using UV-1800 Shimadzu UV–Visible spectrophotometer with a quartz cuvette of 1.0 cm path length. The samples were incubated overnight for studying the interactions between native and IILs treated collagen. 2.6. Circular Dichroism Studies Circular dichroism spectra were recorded to study the influence of native and IILs treated collagen using Jasco-815 spectropolarimeter in
Impedance measurements were carried out using CH Instrumental (USA) electrochemical analyzer CH-model 660 B to assess the effect of native and IILs treated collagen 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 Y ¼ Y 0 þ jY } where, Y′ is the real component describing the energy stored and Y″ is the imaginary component depicting the energy dissipated by the system. 2.9. Optical Micrographs Optical micrographs of native and IILs treated RTT collagen fibres for 24 h was monitored under Aven Inc., Digital Mighty Scope, 1.3 M (Product code: 48708-25, Made in Taiwan) at 10× resolution and the changes occurring in the dimensions of RTT was observed. 3. Results and Discussion 3.1. Viscosity Analysis The rheological behavior for native and IILs treated collagen reflects the strength of its inter-molecular forces. It is said that stronger the inter-molecular forces the higher the viscosity due to its higher shear strength. Molecules get close together and tangled up thereby resisting the flow. Absolute viscosity for native and IILs treated collagen indicates the changes in the inter-molecular and as well as intra-molecular forces of collagen. Decrease in the viscosity of collagen attributes to the aggregation of collagen when treated with higher concentrations of imidazolium-based ILs, 1:10% (Fig. 1). Concentration dependent decrease in the rheology of IILs treated collagen is shown in Supplementary Fig. (S2). The speculated reason behind the drastic decrease in viscosity for C-IHS4 (imidazolium hydrogen sulfate) and C-IS4 (imidazolium sulfate) might be owing to the strong electrostatic interaction, which causes the reorientation in the surrounding water milieu bringing out the huge aggregation effects. There was a notable decrease in rheology of collagen treated with C-IDCA4 (imidazolium dicyanamide) and C-IA4 (imidazolium acetate), whereas slight decrease was witnessed for C-IDP4 (imidazolium dimethyl phosphate) and CIDHP4 (imidazolium dihydrogen phosphate) suggesting changes in the microenvironment of collagen. There are several key factors, which might play a role in altering the rheology of collagen, i.e.
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disturbance around polypeptide chain with the masking of tyrosine residues around 278 nm. Supplementary Fig. S3 also connotes the changes in the absorption spectra after treating collagen with imidazoliumbased ILs (IILs). As seen from S3, there were slight changes in the microenvironment of collagen at lower concentrations (1:0.05, 1:0.5 and 1:5%). There must be plausible interaction between ions and functional groups of collagen and the changes in the molecular interior of collagen might have altered the conformational stability. Therefore, imidazolium based ILs (IILs) prominently affects the solvation environment of collagen, which would evidently reorient the hydration network at its molecular level. 3.3. Circular Dichroism Studies
Fig. 1. Absolute viscosity for native and IILs treated collagen, [C-IDCA4- (Collagen) -(Imidazolium dicyanamide) – (1:10%)]; [C-IHS4- (Collagen) - (Imidazolium hydrogen sulfate) – (1:10%)]; [C-IDP4- (Collagen) – (Imidazolium dimethyl phosphate) – (1:10%)]; [C-IA4- (Collagen) − (Imidazolium acetate) – (1:10%)]; [C-IS4- (Collagen) (Imidazolium sulfate) – (1:10%)]; [C-IDHP4- (Collagen) – (Imidazolium dihydrogen phosphate) – (1:10%)], (Conditions: pH 4; Temperature = 25 °C; Concentration = 0.8 μM).
electrostatic attraction or repulsions, van der Waals forces, hydrogen bonding and hydrophobic interactions [29]. 3.2. UV–Vis Spectroscopic Studies The absorption spectra for native and IILs treated collagen are given in Fig. 2. Collagen has two absorption maxima, the strongest absorption peak around 220 nm discloses the peptide absorption peak, whereas the feeble interaction was observed around 278 nm, which attributes to π–π* electron transition for aromatic amino acid residue (tyrosine), as the tryptophan is absent in type I collagen and the absorbance of phenylalanine is negligible [30]. As seen from Fig. 2, the addition of imidazolium-based ILs (IILs) at higher concentration (1:10%) leads to peak broadening and also the shift of peak at 220 nm suggesting
Fig. 2. UV–Vis absorption studies for native and IILs treated collagen, [C-IDCA4- (Collagen) - (Imidazolium dicyanamide) − (1:10%)]; [C-IHS4- (Collagen) - (Imidazolium hydrogen sulfate) – (1:10%)]; [C-IDP4- (Collagen) - (Imidazolium dimethyl phosphate) – (1:10%)]; [C-IA4- (Collagen) - (Imidazolium acetate) – (1:10%)]; [C-IS4- (Collagen) - (Imidazolium sulfate) – (1:10%)]; [C-IDHP4- (Collagen) - (Imidazolium dihydrogen phosphate) – (1:10%)], (Conditions: pH 4; Temperature = 25 °C; Concentration = 0.8 μM).
Circular dichroic spectroscopic studies have been employed to examine the alterations in the secondary structure of collagen owing to the interaction of imidazolium-based ILs (IILs) by monitoring the changes in the molar ellipticity value at 222 nm. In the far UV region, CD spectrum of type I collagen exhibit peak at 197 nm, which is indicative of absorption region of peptide linkages corresponding to backbone configuration and the peak at 222 nm ascribes polyproline II conformation, which are affirmative to the characteristic secondary structure of collagen [31]. The change in molar ellipticity at positive peak implicates the changes in the polyproline II conformation, albeit negative peak attributes to the conformational changes in the peptide chain. Collagen on denaturation leads to red shift and also the decrease in intensity of negative band, whereas the positive peak around 222 nm disappears. The CD spectra for native and IILs treated collagen (Imidazolium dicyanamide- IDCA4, Imidazolium hydrogen sulfateIHS4, Imidazolium dimethyl phosphate- IDP4, Imidazolium acetateIA4, Imidazolium sulfate- IS4 and Imidazolium dihydrogen phosphateIDHP4) are shown in Fig. 3. It was observed that there was complete disappearance of peaks at 197 and 222 nm for all IILs treated collagen, which indicates the complete aggregation of collagen when treated with imidazolium-based ILs (IILs) at higher percentages (1:10%). Supplementary Fig. S4 connotes the CD spectra of native and IILs treated collagen at different concentration (1:0.05, 1:0.5% and 1:5%). It was seen from S4, at lower concentrations, (1:0.05% and 1:0.5%), there was a slight decrease in molar ellipticity values when compared to native collagen. Concentration dependent decrease in molar ellipticity values
Fig. 3. Changes in the far-UV CD spectra for native and IILs treated collagen, [C-IDCA4(Collagen) - (Imidazolium dicyanamide) – (1:10%)]; [C-IHS4- (Collagen) - (Imidazolium hydrogen sulfate) – (1:10%)]; [C-IDP4- (Collagen) - (Imidazolium dimethyl phosphate) – (1:10%)]; [C-IA4- (Collagen) - (Imidazolium acetate) – (1:10%)]; [C-IS4(Collagen) - (Imidazolium sulfate) – (1:10%)]; [C-IDHP4- (Collagen) - (Imidazolium dihydrogen phosphate) – (1:10%)], (Conditions: pH 4; Temperature = 25 °C; Concentration = 0.8 μM).
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was observed with the increase in concentration of imidazolium-based ILs (IILs). The decrease in the characteristic CD peak intensities of collagen at λmax 222 nm indicates the interaction of IILs with collagen and decreased stability of the collagen attributes to aggregation of collagen molecule at higher IILs concentration. This must be owing to electrostatic interactions between the ions and amino acid residues of protein, which resulted in alteration of secondary structure of collagen. 3.4. FT-IR Spectral Analysis Fourier transform infrared spectroscopy is a rapid and a nondestructive technique to investigate the interactions between the collagen and imidazolium-based ionic liquid (IILs) at the molecular level. Collagen has its characteristic vibration peaks at amide A (N\\H and O\\H stretching at 3400 cm−1), amide B (C\\H stretching at 2930 cm−1), amide I (carbonyl stretching (C_O) in acetamide group of amide groups at 1640 cm−1), amide II (N\\H stretching vibration strongly coupled to the C\\N stretching vibration of collagen amide groups at 1560 cm−1) and amide III (C\\N stretching and N\\H in plane bending from amide linkages at 1240 cm−1) providing information on the secondary structure of collagen [32]. Fig. 4 elucidates the FT-IR spectra for native and IILs treated collagen at higher concentration (Imidazolium dicyanamide- IDCA4, Imidazolium hydrogen sulfateIHS4, Imidazolium dimethyl phosphate- IDP4, Imidazolium acetateIA4, Imidazolium sulfate- IS4 and Imidazolium dihydrogen phosphateIDHP4). Supplementary Fig. S5 exhibits various imidazolium based ILs (IILs) at lower concentration (1:0.05, 1:0.5, 1:5% and neat imidazolium ILs). Characteristic IDCA (imidazolium dicyanamide) peaks were observed at 2131 cm−1, which corresponds to antisymmetric C\\N stretching, 2192 cm−1 accords to symmetric C\\N stretching and 2229 cm−1 attributes to a combination band of symmetric and antisymmetric C\\N stretching mode [33]. The appearance of these bands in CIDCA3 and C-IDCA4 and also the shifts in amide peaks elucidates the
stronger interaction of collagen functional groups with dicyanamide ions at higher concentration, whereas feeble interaction was observed at lower concentrations. The shifts in frequency of amide I and II peaks and the disappearance of amide III bands from C-IDCA3 and C-IDCA4 signifies the change in microenvironment of collagen, when the molecules interact with each other. It was noted that there was complete disappearance of amide A and B peaks for C-IHS3 and C-IHS4 (imidazolium hydrogen sulfate) with the appearance of new peaks around 1301, 1264 and 1161 cm−1, which corresponds to S_O stretching and bending respectively [34]. There was also slight shift in frequencies for amide I, whereas a huge blue shift in peaks was observed for amide II and III around 1548 and 1280 cm−1 respectively, which might be owing to conformational changes in triple helical structure of collagen. Slight shifts in frequencies were observed for C-IDP [1–4] (imidazolium dimethyl phosphate) and C-IA [1–4] (imidazolium acetate) treated collagen with the disappearance of amide III bands, which might be due to feeble interactions between ions and functional groups of collagen. There were negligible shifts in peaks for amide I and II for C-IS, imidazolium sulfate [1 and 2], whereas the witnessing disappearance of amide III bands. For C-IDHP (imidazolium dihydrogen phosphate), amide I remained constant when compared to native collagen, whereas the amide II and III manifested a huge shift and the change in frequencies could be due to the anions, which possibly causes the change in hydration dynamics of collagen surrounding water molecules. This was in coherence with the circular dichroic studies, which confirms the huge aggregation leading to destabilization of collagen at molecular level. IHS (imidazolium hydrogen sulfate) have shown huge destabilizing effect followed by IS (imidazolium sulfate), IDCA (imidazolium dicyanamide) albeit IA (imidazolium acetate), IDP (imidazolium dimethyl phosphate) and IDHP (imidazolium dihydrogen phosphate) have shown lesser effect compared to native collagen and this is suggestive of varying ionic strength. Destabilizing effect on collagen has been exhibited in the following order of Hofmeister series; IDP b IDHP b IA b IDCA b IS b IHS, which is distinct from various spectroscopic studies. 3.5. Dielectric Studies
Fig. 4. FTIR spectra for native and IILs treated collagen, [C-IDCA4- (Collagen) -(Imidazolium dicyanamide) – (1:10%)]; [C-IHS4- (Collagen) - (Imidazolium hydrogen sulfate) – (1:10%)]; [C-IDP4- (Collagen) - (Imidazolium dimethyl phosphate) – (1:10%)]; [C-IA4- (Collagen) - (Imidazolium acetate) – (1:10%)]; [C-IS4- (Collagen) - (Imidazolium sulfate) – (1:10%)]; [C-IDHP4- (Collagen) - (Imidazolium dihydrogen phosphate) – (1:10%)], (Conditions: pH 4; Temperature = 25 °C; Concentration = 0.8 μM).
Dielectric measurement is a profound sensitive tool for studying the dynamics of water surrounding biomolecules. It provides the powerful insights into the hydration behavior of the biomacromolecules. Usually an electric field is applied, it causes the free and bound charges of the biomacromolecules to drift and displace, thus inducing conduction and polarization of currents [35]. The dielectric plot of native and IILs treated collagen (C: IILs- 1:10%) is shown in Fig. 5a and b. Fig. 5a and b elucidates the Nyquist plot (Y′ vs Y″) for studying the admittance and Cole-Cole plot (Z' vs Z") ascertains the total impedance. Supplementary Fig. S6 & S7 exhibits the Nyquist and Cole-Cole plot for various imidazolium based ILs (IILs) at lower concentration (1:0.05, 1:0.5, 1:5% and neat imidazolium ILs). It was seen from the Fig. 5a that collagen has the lowest permittivity, whereas the IILs treated collagen showed the highest permittivity. There might be reorganization in the charges of polar functional groups on the surface of the collagen. IILs treated collagen present at the interface influences this reorganization process, thereby fluctuating between various conformational states and this might be owing to the electrostatic interactions between collagen and IILs [36]. The major factors responsible for degree of hydration includes the molecular asymmetry, polar nature, the hydrogen bonding capacity and the strength of forming ion pairs between the amino and carboxyl group of protein and other polar functional groups of IILs. It is hypothesized that there will be a strong electrostatic interaction with charged residues like glutamate and lysine residues. Since, the collagen is constituted by polar repeating units of amino acids and ionic liquids; there will be forced interactions between these molecules, which lead to the exposure of hydrophobic patches and the burial of hydrophilic groups into the core. Usually, the hydrophobic groups of protein molecule are buried into the interior core whereas the hydrophilic groups reside on
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Fig. 5. Dielectric spectra for native and IILs treated collagen, a. Nyquist Plot; b. Bode-Bode plot; [C-IDCA4- (Collagen) -(Imidazolium dicyanamide) – (1:10%)]; [C-IHS4- (Collagen) (Imidazolium hydrogen sulfate) – (1:10%)]; [C-IDP4- (Collagen) - (Imidazolium dimethyl phosphate) – (1:10%)]; [C-IA4- (Collagen) - (Imidazolium acetate) – (1:10%)]; [C-IS4(Collagen) - (Imidazolium sulfate) – (1:10%)]; [C-IDHP4- (Collagen) - (Imidazolium dihydrogen phosphate) – (1:10%)], (Conditions: pH 4; Temperature = 25 °C; Concentration = 0.8 μM).
the surface of protein, which has an affinity to interact with water molecules from its surrounding aqueous environment. Addition of IILs resulted in decrease in phase angle when compared to native collagen (Fig. 5b). This might be owing to the collagen is a charged molecule and addition of ILs leads to its charged behavior. The hydration water also plays a crucial role in maintaining the stability of biomacromolecules. Water molecules surrounding protein will have different interactions depending on the hydration shell. First layer corresponds to the molecules in the immediate vicinity of protein, second and third layer corresponding to loosely bound molecules, which interact with the tightly bound water and surrounding bulk water, which is considered to be unaffected by surface of the protein. It is speculated that the chaotropic anion will have stronger interactions with hydration shell thereby exposing the hydrophobic patches and the burial of hydrophilic groups into interior core of collagen, thereby leading to the destabilization of collagen at higher concentration.
rat tail tendon collagen fibres retains the helicity of the fibrils and the microscopic banding pattern when treated with water [37]. IDCA (imidazolium dicyanamide), IDP (imidazolium dimethyl phosphate) and IA (imidazolium acetate) at highest concentration (10%) have shown slight swelling, albeit IHS (imidazolium hydrogen sulfate), IS (imidazolium sulfate) and IDHP (imidazolium dihydrogen phosphate) witnessed huge distortion at highest concentration, which might be owing to the chaotropicity of ions and reorientation in the surrounding water milieu leading to the agglomeration. Imidazolium cations along with varying anions have shown swelling effect in this order (IDP b IA b IDCA b IHS b IS b IDHP). This might be due to the ionic strength, better the strength higher the swelling, which have led to distorted wave pattern. Supplementary Fig. S8 unveils concentration dependent swelling when treated with imidazolium based ILs (IILs).
3.6. Dimensional Stability for Native and RTT Treated with Imidazolium Based ILs
Collagen treated with varying imidazolium-based ionic liquids (IILs) was studied at molecular and fibrillar level. It was found that the viscosity, UV–Vis, impedance, circular dichroic and FT-IR studies show denaturation at higher concentration, whereas slight conformational changes was observed at lower concentration resulting in structural disorientation of collagen. Increased dimensions in RTT collagen fibres
Rat tail tendon collagen fibres treated with water and various imidazolium based ILs for 24 h at 25 °C, revealing its impact on dimensional stability of RTT collagen fibres (Fig. 6). It was evidenced that the
4. Conclusions
Fig. 6. Optical micrographs for RTT treated with a. water; b. IDCA- Imidazolium dicyanamide- 10%; c. IHS- Imidazolium hydrogen sulfate- 10%; d. IDP- Imidazolium dimethyl phosphate10%; e. IA- Imidazolium acetate- 10%; f. IS- Imidazolium sulfate- 10% and g. IDHP- Imidazolium dihydrogen phosphate- 10%; (Conditions: Temperature = 25 °C; Incubation time = 24 h).
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treated with imidazolium-based ILs (IILs) was descried indicating distortions in wave patterns. The speculation behind the observed structural deformation is the strong electrostatic interaction between anions of imidazolium-based ILs (IILs) to the functional groups of collagen. This might be due to the molecular reorientation in the surrounding water milieu leading to the structural disorientation. It is well known that the combination of kosmotropic cation and chaotropic anion destabilizes collagen as the chaotropes are found to be poorly hydrated, allowing them to preferentially interact with the protein rather than the water. As they are said to be structure breakers, macromolecules have more structural freedom for extension and denaturation. Imidazolium cations with varied anions have demonstrated the destabilizing effect on collagen in the following order of Hofmeister series; IDP b IDHP b IA b IDCA b IS b IHS, at both molecular and fibrillar levels. Concerning this effect, further investigations and optimizations for various applications are under evaluation. Abbreviations IILs RTT CD FT-IR
Imidazolium-based ionic liquids rat tail tendons circular dichroism Fourier transform infrared spectroscopy
Acknowledgment One of the authors, AT acknowledges CSIR for Senior Research Fellowship. CLRI communication code: 1300. Appendix A. Supplementary data This study proves the influence of varying anions on imidazoliumbased ILs (IILs) through various experimental studies. Supplementary data to this article can be found online at doi:https://doi.org/10.1016/j. saa.2019.01.029. References [1] K. Fujita, D.R. MacFarlane, M. Forsyth, Protein Solubilising and stabilizing ionic liquids, Chem. Commun. (38) (2005) 4804–4806. [2] S. Pandey, Analytical applications of room-temperature ionic liquids: a review of recent efforts, Anal. Chim. Acta 556 (2006) 38–45. [3] D. Wei, A. Ivaska, Application of ionic liquid in ionic liquid sensors, Anal. Chim. Acta 607 (2008) 126–135. [4] T. Ueki, M. Watanabe, Macromolecules in ionic liquids: Progress, challenges, and opportunities, Macromolecules 41 (2008) 3739–3749. [5] Y. Zhang, P.S. Cremer, Interactions between macromolecules and ions: the Hofmeister series, Curr. Opin. Chem. Biol. 10 (2006) 1–6. [6] Y. Shu, M. Liu, S. Chen, J. Wang, New insight into molecular interactions of imidazolium ionic liquids with bovine serum albumin, J. Phys. Chem. B 115 (2011) 12306–12314. [7] L.Y. Zhu, G.Q. Li, F.Y. Zheng, Interaction of bovine serum albumin with two Alkylimidazolium-based ionic liquids investigated by microcalorimetry and circular dichroism, Biophys. Chem. 2 (2011) 146–151. [8] H. Yan, J. Wu, G. Dai, A. Zhong, H. Chen, J. Yang, D. Han, Interaction mechanism of ionic liquids [Cnmim] Br (n = 4, 6, 8, 10) with bovine serum albumin, J. Lumin. 132 (2012) 622–628. [9] I. Jha, A. Kumar, P. Venkatesu, The overriding roles of concentration and hydrophobic effect on structure and stability of Heme protein induced by imidazolium –based ionic liquids, J. Phys. Chem. B 119 (2015) 8357–8368.
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Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Stability of collagen in ionic liquids: Ion specific Hofmeister series effect Aafiya Tarannum, Raghava Rao Jonnalagadda, Nishad Fathima Nishter ⁎ Inorganic and Physical Chemistry Laboratory, CSIR-Central Leather Research Institute, Adyar, Chennai-20, India
a r t i c l e
i n f o
Article history: Received 24 October 2018 Received in revised form 4 January 2019 Accepted 15 January 2019 Available online 16 January 2019 Keywords: Imidazolium-based ionic liquid Destabilization RTT collagen fibres Circular dichroism studies Collagen
a b s t r a c t In protein-ionic liquids (ILs) interactions, anions play an important role. In this work, imidazolium-based ILs (IILs) with varying anions namely dicyanamide (DCA), hydrogen sulfate (HS), dimethyl phosphate (DP), acetate (A), sulfate (S) and dihydrogen phosphate (DHP) have been chosen with the aim of understanding the role of anions in bringing about the destabilization effect on collagen based on the kosmotropicity and chaotropicity of ions. Imidazolium-based ILs destabilized the triple helical structure of collagen, thereby proving as strong denaturants for collagen and this was confirmed by various spectroscopic techniques viz., CD, FT-IR, viscosity and impedance measurements. The solution studies were in accordance to the changes in the dimensional stability of RTT collagen fibres at the fibrillar level. Imidazolium cations with varied anions have exhibited destabilizing effect on collagen in order of ions in Hofmeister series; IDP b IDHP b IA b IDCA b IS b IHS. Presumably, these notable effect and changes were facilitated by electrostatic interactions between the anions and amine functional groups of collagen. © 2019 Elsevier B.V. All rights reserved.
1. Introduction Ionic liquids, lime lighted as “green” and “designer” materials have instigated a paradigm shift in every field due to its unique and attractive physicochemical properties [1–2]. Depending on the cations and anions, they can be specifically and effectively tuned for a wide range of applications in green chemistry to pharmaceutical and biotechnological applications [3–4]. In general, a pair of strong kosmotropic anions and chaotropic cations stabilize the protein, whereas chaotropic anions and kosmotropic anions destabilize the protein and selection of ions are based on Hofmeister series [5]. Research on the interaction and behavior between ionic liquid and protein has nested stance in scientific community due to its remarkable properties. The spectral and thermodynamic behavior of IL-BSA system suggested the loss of secondary and tertiary structure and the anionic moiety seemed to have more obvious effect than the cationic ones [6] and the denaturation was caused by electrostatic and hydrophobic interaction, hydrogen bonding and van der Waals forces as proved by Zhu and Yan et al. [7–8]. The stability of insulin and heme protein in imidazolium based ILs with varying anions was also evaluated and it was disappointing to note that ILs failed to protect the native state, which led to denaturation [9–10]. Therefore, protein stability is receiving rapid interest in the active research in biological and physical sciences providing an adequate and fundamental understanding of the behavior and stability of proteins in ILs.
⁎ Corresponding author. E-mail address: [email protected] (N.F. Nishter).
https://doi.org/10.1016/j.saa.2019.01.029 1386-1425/© 2019 Elsevier B.V. All rights reserved.
Collagen, the most abundant protein in mammals has been used widely as a biomaterial [11–12] and they have been explored with ionic liquids [13–14]. The dissolution and regeneration of collagen using BMIM Cl studied by Meng et al. reported partial destruction in triple helical structure [15]. Zhang et al., has prepared cellulose/collagen film using [EMIM] [Ac] as common solvent [16] and they have been used in regenerating triple helical structure, which is indicative of potential applications in tissue engineering and it has been proved using experimental and computational studies [17–18]. We have studied the influence of various ionic liquids on collagen on the different hierarchical orderings of collagen. The influence of BMIM Cl has brought significant changes at higher hierarchical level of collagen [19]. At molecular and fibrillar level, choline amino acid based ILs has also demonstrated destabilizing effect due to competitive hydrogen bonding between its molecules [20]. Also, ammonium and phosphonium based ILs had demonstrated destabilizing effect on collagen due to their chaotropicity of ions [21–22], whilst choline dihydrogen phosphate stabilized collagen by exerting an electrostatic force, thus making it suitable for preparation of biomaterials for tissue engineering applications [23–24]. Also, the changes in the structure and hydration dynamics of collagen has been studied upon interaction with imidazolium and choline based ILs [25]. In addition to, imidazolium based ILs has been employed as unhairing cum fibre agent for skin matrix and it has paved way for cleaner and greener processing systems [26–27]. Still the interactions between proteins and ionic liquids in aqueous media are not well understood and the underlying interpretations are mostly speculative. This emphasizes the need to understand the interaction of collagen with various additives, thus providing a new probe for elucidating protein- IL interaction. Herein, a series of imidazolium-
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based ionic liquid (IILs) with wide range of varying anions such as dicyanamide, hydrogen sulfate, dimethyl phosphate, acetate, sulfate and dihydrogen phosphate has been comprehensively investigated at both molecular and fibrillar level. The choice of cations and anions was based on chaotropic and kosmotropic behavior of ions as described in Hofmeister series. The interaction of IILs with collagen has been studied using various characterization techniques viz., circular dichroic studies, Fourier transform infrared spectroscopy, viscosity and UV–Vis studies, dimensional stability and impedance measurements to influence the varying anions on collagen. 2. Experimental
the far-UV region ranging from 260 to 190 nm and it was scanned with 0.2 nm intervals with a path length 1 mm at 25 °C with computer averaged three scans for each sample. The data were obtained in milli degrees and further they are converted to molar ellipticity (deg·cm2·dmol−1). Molar ellipticity was plotted against wavelength in nanometers (nm). 2.7. FT-IR Studies Lyophilized samples of native and IILs treated collagen were studied using Jasco FT-IR-4200 (Fourier Transform Infrared Spectrometer) with 40 scans in the range of 4000–400 cm−1 and the resolution of 4 cm−1. The studies were carried out using KBr pellet method.
2.1. Materials 2.8. Impedance Measurements 1-Butyl-3-methylimidazolium dicyanamide (IDCA), 1-Butyl-3methylimidazolium hydrogen sulfate (IHS), 1-Butyl-3methylimidazolium dimethyl phosphate (IDP), 1-Butyl-3methylimidazolium acetate (IA), 1-Butyl-3-methylimidazolium sulfate (IS) and 1-Butyl-3-methylimidazolium dihydrogen phosphate (IDHP) were procured from Sigma Aldrich and Ionic Liquid Technologies GmbH (IoLiTec, Germany). Type I collagen was extracted from sixth month old albino rats. 2.2. Extraction of Type I Collagen Rat tail tendons were used for extracting the type I collagen. RTT were teased and washed thoroughly with 0.9% saline at 4 °C. Purification was carried out using 5% sodium chloride and centrifuged for garnering the precipitates. The precipitate obtained was redissolved in 0.5 M acetic acid and dialyzed against 50 mM phosphate buffer with pH of 6.5 ± 0.2. Then the precipitated collagen was re-dissolved in 0.5 M acetic acid with the final dialysis against 0.05 M acetic acid. Hydroxyproline content was done to estimate the concentration of collagen using method of Woessner [28]. SDS-PAGE was carried out to confirm the purity of collagen (Supplementary Fig. S1). The obtained collagen solution was stored at 4 °C. 2.3. Preparation of Collagen IILs Solution The collagen solution in acetate buffer was treated with varying concentrations of IILs. IDCA, IA, IDP, IHS, IS, IDHP [1–4] was stirred continuously at 4 °C for 3 h. The concentration are as follows; C-I1–1: 0.05%, C\\I2\\ (1:0.5%), C\\I3\\ (1: 5%), C\\I4\\ (1:10%) throughout to prevent denaturation of collagen due to heat generated during mixing. The working concentration of collagen was taken as 0.8 μM at pH 4.0. The concentration of collagen was kept constant and IILs concentration was varied. 2.4. Viscosity Measurements Viscosity measurements were carried out to study the influence of native and IILs treated collagen on the rheological property using a Brookfield DV-II + Pro viscometer at 25 °C. 2.5. UV–Vis Studies Native and IILs treated collagen was appraised using UV-1800 Shimadzu UV–Visible spectrophotometer with a quartz cuvette of 1.0 cm path length. The samples were incubated overnight for studying the interactions between native and IILs treated collagen. 2.6. Circular Dichroism Studies Circular dichroism spectra were recorded to study the influence of native and IILs treated collagen using Jasco-815 spectropolarimeter in
Impedance measurements were carried out using CH Instrumental (USA) electrochemical analyzer CH-model 660 B to assess the effect of native and IILs treated collagen 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 Y ¼ Y 0 þ jY } where, Y′ is the real component describing the energy stored and Y″ is the imaginary component depicting the energy dissipated by the system. 2.9. Optical Micrographs Optical micrographs of native and IILs treated RTT collagen fibres for 24 h was monitored under Aven Inc., Digital Mighty Scope, 1.3 M (Product code: 48708-25, Made in Taiwan) at 10× resolution and the changes occurring in the dimensions of RTT was observed. 3. Results and Discussion 3.1. Viscosity Analysis The rheological behavior for native and IILs treated collagen reflects the strength of its inter-molecular forces. It is said that stronger the inter-molecular forces the higher the viscosity due to its higher shear strength. Molecules get close together and tangled up thereby resisting the flow. Absolute viscosity for native and IILs treated collagen indicates the changes in the inter-molecular and as well as intra-molecular forces of collagen. Decrease in the viscosity of collagen attributes to the aggregation of collagen when treated with higher concentrations of imidazolium-based ILs, 1:10% (Fig. 1). Concentration dependent decrease in the rheology of IILs treated collagen is shown in Supplementary Fig. (S2). The speculated reason behind the drastic decrease in viscosity for C-IHS4 (imidazolium hydrogen sulfate) and C-IS4 (imidazolium sulfate) might be owing to the strong electrostatic interaction, which causes the reorientation in the surrounding water milieu bringing out the huge aggregation effects. There was a notable decrease in rheology of collagen treated with C-IDCA4 (imidazolium dicyanamide) and C-IA4 (imidazolium acetate), whereas slight decrease was witnessed for C-IDP4 (imidazolium dimethyl phosphate) and CIDHP4 (imidazolium dihydrogen phosphate) suggesting changes in the microenvironment of collagen. There are several key factors, which might play a role in altering the rheology of collagen, i.e.
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disturbance around polypeptide chain with the masking of tyrosine residues around 278 nm. Supplementary Fig. S3 also connotes the changes in the absorption spectra after treating collagen with imidazoliumbased ILs (IILs). As seen from S3, there were slight changes in the microenvironment of collagen at lower concentrations (1:0.05, 1:0.5 and 1:5%). There must be plausible interaction between ions and functional groups of collagen and the changes in the molecular interior of collagen might have altered the conformational stability. Therefore, imidazolium based ILs (IILs) prominently affects the solvation environment of collagen, which would evidently reorient the hydration network at its molecular level. 3.3. Circular Dichroism Studies
Fig. 1. Absolute viscosity for native and IILs treated collagen, [C-IDCA4- (Collagen) -(Imidazolium dicyanamide) – (1:10%)]; [C-IHS4- (Collagen) - (Imidazolium hydrogen sulfate) – (1:10%)]; [C-IDP4- (Collagen) – (Imidazolium dimethyl phosphate) – (1:10%)]; [C-IA4- (Collagen) − (Imidazolium acetate) – (1:10%)]; [C-IS4- (Collagen) (Imidazolium sulfate) – (1:10%)]; [C-IDHP4- (Collagen) – (Imidazolium dihydrogen phosphate) – (1:10%)], (Conditions: pH 4; Temperature = 25 °C; Concentration = 0.8 μM).
electrostatic attraction or repulsions, van der Waals forces, hydrogen bonding and hydrophobic interactions [29]. 3.2. UV–Vis Spectroscopic Studies The absorption spectra for native and IILs treated collagen are given in Fig. 2. Collagen has two absorption maxima, the strongest absorption peak around 220 nm discloses the peptide absorption peak, whereas the feeble interaction was observed around 278 nm, which attributes to π–π* electron transition for aromatic amino acid residue (tyrosine), as the tryptophan is absent in type I collagen and the absorbance of phenylalanine is negligible [30]. As seen from Fig. 2, the addition of imidazolium-based ILs (IILs) at higher concentration (1:10%) leads to peak broadening and also the shift of peak at 220 nm suggesting
Fig. 2. UV–Vis absorption studies for native and IILs treated collagen, [C-IDCA4- (Collagen) - (Imidazolium dicyanamide) − (1:10%)]; [C-IHS4- (Collagen) - (Imidazolium hydrogen sulfate) – (1:10%)]; [C-IDP4- (Collagen) - (Imidazolium dimethyl phosphate) – (1:10%)]; [C-IA4- (Collagen) - (Imidazolium acetate) – (1:10%)]; [C-IS4- (Collagen) - (Imidazolium sulfate) – (1:10%)]; [C-IDHP4- (Collagen) - (Imidazolium dihydrogen phosphate) – (1:10%)], (Conditions: pH 4; Temperature = 25 °C; Concentration = 0.8 μM).
Circular dichroic spectroscopic studies have been employed to examine the alterations in the secondary structure of collagen owing to the interaction of imidazolium-based ILs (IILs) by monitoring the changes in the molar ellipticity value at 222 nm. In the far UV region, CD spectrum of type I collagen exhibit peak at 197 nm, which is indicative of absorption region of peptide linkages corresponding to backbone configuration and the peak at 222 nm ascribes polyproline II conformation, which are affirmative to the characteristic secondary structure of collagen [31]. The change in molar ellipticity at positive peak implicates the changes in the polyproline II conformation, albeit negative peak attributes to the conformational changes in the peptide chain. Collagen on denaturation leads to red shift and also the decrease in intensity of negative band, whereas the positive peak around 222 nm disappears. The CD spectra for native and IILs treated collagen (Imidazolium dicyanamide- IDCA4, Imidazolium hydrogen sulfateIHS4, Imidazolium dimethyl phosphate- IDP4, Imidazolium acetateIA4, Imidazolium sulfate- IS4 and Imidazolium dihydrogen phosphateIDHP4) are shown in Fig. 3. It was observed that there was complete disappearance of peaks at 197 and 222 nm for all IILs treated collagen, which indicates the complete aggregation of collagen when treated with imidazolium-based ILs (IILs) at higher percentages (1:10%). Supplementary Fig. S4 connotes the CD spectra of native and IILs treated collagen at different concentration (1:0.05, 1:0.5% and 1:5%). It was seen from S4, at lower concentrations, (1:0.05% and 1:0.5%), there was a slight decrease in molar ellipticity values when compared to native collagen. Concentration dependent decrease in molar ellipticity values
Fig. 3. Changes in the far-UV CD spectra for native and IILs treated collagen, [C-IDCA4(Collagen) - (Imidazolium dicyanamide) – (1:10%)]; [C-IHS4- (Collagen) - (Imidazolium hydrogen sulfate) – (1:10%)]; [C-IDP4- (Collagen) - (Imidazolium dimethyl phosphate) – (1:10%)]; [C-IA4- (Collagen) - (Imidazolium acetate) – (1:10%)]; [C-IS4(Collagen) - (Imidazolium sulfate) – (1:10%)]; [C-IDHP4- (Collagen) - (Imidazolium dihydrogen phosphate) – (1:10%)], (Conditions: pH 4; Temperature = 25 °C; Concentration = 0.8 μM).
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was observed with the increase in concentration of imidazolium-based ILs (IILs). The decrease in the characteristic CD peak intensities of collagen at λmax 222 nm indicates the interaction of IILs with collagen and decreased stability of the collagen attributes to aggregation of collagen molecule at higher IILs concentration. This must be owing to electrostatic interactions between the ions and amino acid residues of protein, which resulted in alteration of secondary structure of collagen. 3.4. FT-IR Spectral Analysis Fourier transform infrared spectroscopy is a rapid and a nondestructive technique to investigate the interactions between the collagen and imidazolium-based ionic liquid (IILs) at the molecular level. Collagen has its characteristic vibration peaks at amide A (N\\H and O\\H stretching at 3400 cm−1), amide B (C\\H stretching at 2930 cm−1), amide I (carbonyl stretching (C_O) in acetamide group of amide groups at 1640 cm−1), amide II (N\\H stretching vibration strongly coupled to the C\\N stretching vibration of collagen amide groups at 1560 cm−1) and amide III (C\\N stretching and N\\H in plane bending from amide linkages at 1240 cm−1) providing information on the secondary structure of collagen [32]. Fig. 4 elucidates the FT-IR spectra for native and IILs treated collagen at higher concentration (Imidazolium dicyanamide- IDCA4, Imidazolium hydrogen sulfateIHS4, Imidazolium dimethyl phosphate- IDP4, Imidazolium acetateIA4, Imidazolium sulfate- IS4 and Imidazolium dihydrogen phosphateIDHP4). Supplementary Fig. S5 exhibits various imidazolium based ILs (IILs) at lower concentration (1:0.05, 1:0.5, 1:5% and neat imidazolium ILs). Characteristic IDCA (imidazolium dicyanamide) peaks were observed at 2131 cm−1, which corresponds to antisymmetric C\\N stretching, 2192 cm−1 accords to symmetric C\\N stretching and 2229 cm−1 attributes to a combination band of symmetric and antisymmetric C\\N stretching mode [33]. The appearance of these bands in CIDCA3 and C-IDCA4 and also the shifts in amide peaks elucidates the
stronger interaction of collagen functional groups with dicyanamide ions at higher concentration, whereas feeble interaction was observed at lower concentrations. The shifts in frequency of amide I and II peaks and the disappearance of amide III bands from C-IDCA3 and C-IDCA4 signifies the change in microenvironment of collagen, when the molecules interact with each other. It was noted that there was complete disappearance of amide A and B peaks for C-IHS3 and C-IHS4 (imidazolium hydrogen sulfate) with the appearance of new peaks around 1301, 1264 and 1161 cm−1, which corresponds to S_O stretching and bending respectively [34]. There was also slight shift in frequencies for amide I, whereas a huge blue shift in peaks was observed for amide II and III around 1548 and 1280 cm−1 respectively, which might be owing to conformational changes in triple helical structure of collagen. Slight shifts in frequencies were observed for C-IDP [1–4] (imidazolium dimethyl phosphate) and C-IA [1–4] (imidazolium acetate) treated collagen with the disappearance of amide III bands, which might be due to feeble interactions between ions and functional groups of collagen. There were negligible shifts in peaks for amide I and II for C-IS, imidazolium sulfate [1 and 2], whereas the witnessing disappearance of amide III bands. For C-IDHP (imidazolium dihydrogen phosphate), amide I remained constant when compared to native collagen, whereas the amide II and III manifested a huge shift and the change in frequencies could be due to the anions, which possibly causes the change in hydration dynamics of collagen surrounding water molecules. This was in coherence with the circular dichroic studies, which confirms the huge aggregation leading to destabilization of collagen at molecular level. IHS (imidazolium hydrogen sulfate) have shown huge destabilizing effect followed by IS (imidazolium sulfate), IDCA (imidazolium dicyanamide) albeit IA (imidazolium acetate), IDP (imidazolium dimethyl phosphate) and IDHP (imidazolium dihydrogen phosphate) have shown lesser effect compared to native collagen and this is suggestive of varying ionic strength. Destabilizing effect on collagen has been exhibited in the following order of Hofmeister series; IDP b IDHP b IA b IDCA b IS b IHS, which is distinct from various spectroscopic studies. 3.5. Dielectric Studies
Fig. 4. FTIR spectra for native and IILs treated collagen, [C-IDCA4- (Collagen) -(Imidazolium dicyanamide) – (1:10%)]; [C-IHS4- (Collagen) - (Imidazolium hydrogen sulfate) – (1:10%)]; [C-IDP4- (Collagen) - (Imidazolium dimethyl phosphate) – (1:10%)]; [C-IA4- (Collagen) - (Imidazolium acetate) – (1:10%)]; [C-IS4- (Collagen) - (Imidazolium sulfate) – (1:10%)]; [C-IDHP4- (Collagen) - (Imidazolium dihydrogen phosphate) – (1:10%)], (Conditions: pH 4; Temperature = 25 °C; Concentration = 0.8 μM).
Dielectric measurement is a profound sensitive tool for studying the dynamics of water surrounding biomolecules. It provides the powerful insights into the hydration behavior of the biomacromolecules. Usually an electric field is applied, it causes the free and bound charges of the biomacromolecules to drift and displace, thus inducing conduction and polarization of currents [35]. The dielectric plot of native and IILs treated collagen (C: IILs- 1:10%) is shown in Fig. 5a and b. Fig. 5a and b elucidates the Nyquist plot (Y′ vs Y″) for studying the admittance and Cole-Cole plot (Z' vs Z") ascertains the total impedance. Supplementary Fig. S6 & S7 exhibits the Nyquist and Cole-Cole plot for various imidazolium based ILs (IILs) at lower concentration (1:0.05, 1:0.5, 1:5% and neat imidazolium ILs). It was seen from the Fig. 5a that collagen has the lowest permittivity, whereas the IILs treated collagen showed the highest permittivity. There might be reorganization in the charges of polar functional groups on the surface of the collagen. IILs treated collagen present at the interface influences this reorganization process, thereby fluctuating between various conformational states and this might be owing to the electrostatic interactions between collagen and IILs [36]. The major factors responsible for degree of hydration includes the molecular asymmetry, polar nature, the hydrogen bonding capacity and the strength of forming ion pairs between the amino and carboxyl group of protein and other polar functional groups of IILs. It is hypothesized that there will be a strong electrostatic interaction with charged residues like glutamate and lysine residues. Since, the collagen is constituted by polar repeating units of amino acids and ionic liquids; there will be forced interactions between these molecules, which lead to the exposure of hydrophobic patches and the burial of hydrophilic groups into the core. Usually, the hydrophobic groups of protein molecule are buried into the interior core whereas the hydrophilic groups reside on
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Fig. 5. Dielectric spectra for native and IILs treated collagen, a. Nyquist Plot; b. Bode-Bode plot; [C-IDCA4- (Collagen) -(Imidazolium dicyanamide) – (1:10%)]; [C-IHS4- (Collagen) (Imidazolium hydrogen sulfate) – (1:10%)]; [C-IDP4- (Collagen) - (Imidazolium dimethyl phosphate) – (1:10%)]; [C-IA4- (Collagen) - (Imidazolium acetate) – (1:10%)]; [C-IS4(Collagen) - (Imidazolium sulfate) – (1:10%)]; [C-IDHP4- (Collagen) - (Imidazolium dihydrogen phosphate) – (1:10%)], (Conditions: pH 4; Temperature = 25 °C; Concentration = 0.8 μM).
the surface of protein, which has an affinity to interact with water molecules from its surrounding aqueous environment. Addition of IILs resulted in decrease in phase angle when compared to native collagen (Fig. 5b). This might be owing to the collagen is a charged molecule and addition of ILs leads to its charged behavior. The hydration water also plays a crucial role in maintaining the stability of biomacromolecules. Water molecules surrounding protein will have different interactions depending on the hydration shell. First layer corresponds to the molecules in the immediate vicinity of protein, second and third layer corresponding to loosely bound molecules, which interact with the tightly bound water and surrounding bulk water, which is considered to be unaffected by surface of the protein. It is speculated that the chaotropic anion will have stronger interactions with hydration shell thereby exposing the hydrophobic patches and the burial of hydrophilic groups into interior core of collagen, thereby leading to the destabilization of collagen at higher concentration.
rat tail tendon collagen fibres retains the helicity of the fibrils and the microscopic banding pattern when treated with water [37]. IDCA (imidazolium dicyanamide), IDP (imidazolium dimethyl phosphate) and IA (imidazolium acetate) at highest concentration (10%) have shown slight swelling, albeit IHS (imidazolium hydrogen sulfate), IS (imidazolium sulfate) and IDHP (imidazolium dihydrogen phosphate) witnessed huge distortion at highest concentration, which might be owing to the chaotropicity of ions and reorientation in the surrounding water milieu leading to the agglomeration. Imidazolium cations along with varying anions have shown swelling effect in this order (IDP b IA b IDCA b IHS b IS b IDHP). This might be due to the ionic strength, better the strength higher the swelling, which have led to distorted wave pattern. Supplementary Fig. S8 unveils concentration dependent swelling when treated with imidazolium based ILs (IILs).
3.6. Dimensional Stability for Native and RTT Treated with Imidazolium Based ILs
Collagen treated with varying imidazolium-based ionic liquids (IILs) was studied at molecular and fibrillar level. It was found that the viscosity, UV–Vis, impedance, circular dichroic and FT-IR studies show denaturation at higher concentration, whereas slight conformational changes was observed at lower concentration resulting in structural disorientation of collagen. Increased dimensions in RTT collagen fibres
Rat tail tendon collagen fibres treated with water and various imidazolium based ILs for 24 h at 25 °C, revealing its impact on dimensional stability of RTT collagen fibres (Fig. 6). It was evidenced that the
4. Conclusions
Fig. 6. Optical micrographs for RTT treated with a. water; b. IDCA- Imidazolium dicyanamide- 10%; c. IHS- Imidazolium hydrogen sulfate- 10%; d. IDP- Imidazolium dimethyl phosphate10%; e. IA- Imidazolium acetate- 10%; f. IS- Imidazolium sulfate- 10% and g. IDHP- Imidazolium dihydrogen phosphate- 10%; (Conditions: Temperature = 25 °C; Incubation time = 24 h).
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treated with imidazolium-based ILs (IILs) was descried indicating distortions in wave patterns. The speculation behind the observed structural deformation is the strong electrostatic interaction between anions of imidazolium-based ILs (IILs) to the functional groups of collagen. This might be due to the molecular reorientation in the surrounding water milieu leading to the structural disorientation. It is well known that the combination of kosmotropic cation and chaotropic anion destabilizes collagen as the chaotropes are found to be poorly hydrated, allowing them to preferentially interact with the protein rather than the water. As they are said to be structure breakers, macromolecules have more structural freedom for extension and denaturation. Imidazolium cations with varied anions have demonstrated the destabilizing effect on collagen in the following order of Hofmeister series; IDP b IDHP b IA b IDCA b IS b IHS, at both molecular and fibrillar levels. Concerning this effect, further investigations and optimizations for various applications are under evaluation. Abbreviations IILs RTT CD FT-IR
Imidazolium-based ionic liquids rat tail tendons circular dichroism Fourier transform infrared spectroscopy
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