- Email: [email protected]
Effect of chromium(III) gallate complex on stabilization of collagen
International Journal of Biological Macromolecules 96 (2017) 429–435
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Effect of chromium(III) gallate complex on stabilization of collagen Dhanya Jaikumar, Babu Baskaran, V.G. Vaidyanathan ∗ Advanced Materials Laboratory and Academy of Scientific and Innovative Research (AcSIR), CSIR-Central Leather Research Institute, Adyar, Chennai 600 020, India
a r t i c l e
i n f o
Article history: Received 6 October 2016 Received in revised form 16 December 2016 Accepted 17 December 2016 Available online 21 December 2016 Keywords: Collagen Chromium (III) Crosslinking Fibrils Viscosity Polyphenols
a b s t r a c t To improve the stability of the collagen, here we studied the interaction of chromium(III) polyphenolic complex, [Cr(GA)2 ] (GA: Gallic acid) with collagen using various spectroscopic techniques. Circular dichroism studies show that the [Cr(GA)2 ] and gallic acid did not induce any structural perturbations on the triple helix of the collagen. Both differential scanning calorimetric(DSC) data and micro-shrinkage temperature studies showed that [Cr(GA)2 ] stabilized the collagen by 6 ± 1 ◦ C compared to gallic acid. Hydrodynamic studies revealed that the viscosity of collagen drastically reduced in the presence of [Cr(GA)2 ] while gallic acid did not. Fibrillation assay displayed a significant delay in fibril formation with Cr(III) complex compared to gallic acid treated collagen. The inhibition of fibril formation was further confirmed by microscopic data in which collagen fibres are seen with GA while [Cr(GA)2 ] treated collagen exhibit a thin microfibrils. From AFM studies, the d-periodicity of collagen was found to be decreased with [Cr(GA)2 ] while increased with gallic acid. The present study deliberate the advantage of metal complex containing polyphenolic ligand as crosslinking agent due to its synergistic effect of both metal center as well as polyphenolic groups in the stabilization of collagen structure. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Collagen is an essential structural protein as well an important component of extracellular matrix. It accounts for 30% of the total protein content of the body helping in cell adhesion, its proliferation, migration, etc. There are 28 fibrillar and non-fibrillar types of collagen identified to date [1]. Of which, usually the structure of Type I collagen, its hierarchical ordering and self-assembly processes are highlighted. Each alpha chain is a left-handed polyproline intertwined to form a right-handed triple helix. The triple helical structure of collagen composed of three individual alpha chains with Gly-X-Y as repeated sequence observed throughout the length of the chain [2]. Moreover, structure is stabilized by hydrogen bonds with intra- and intermolecular forces. Due to its biodegradability and poor mechanical stability, collagenase attacks the native collagen at physiological pH, temperature and ionic strength easily [3,4]. To improve the mechanical stability and minimize the biodegradation rate, crosslinking of collagen is an essential step. A number of studies have been reported on stabilization of type I collagen by small molecules [5]. Specifically, transition
∗ Corresponding author at: Advanced Materials Laboratory, CSIR-Central Leather Research Institute, Adyar, Chennai 600020, India. E-mail address: [email protected] (V.G. Vaidyanathan). http://dx.doi.org/10.1016/j.ijbiomac.2016.12.049 0141-8130/© 2016 Elsevier B.V. All rights reserved.
metal based complexes are known to bind with collagen and enhance the stability. In particular, chromium with an oxidation state of +3 is preferred over +6 due to its lesser carcinogenicity and binds with aspartic and glutamic residues of collagen by aqua ligand substitution [6]. Similarly, studies on the interaction of polyphenolic compounds with proteins have also been reported for its better stability [7–9]. In addition, it has diverse applications in the pharmaceutical industries. Epigallocatechingallate (EGCG), catechin and gallic acid etc., has been reported to form strong complexes with the proteins [10]. As polyphenolic compounds possess aromatic, hydroxyl and acid groups, it can bind to the proteins through four key interactions i.e., non-covalent, covalent, ionic and H-bonds [11–13]. Krishnamoorthy et al., demonstrated the increased mechanical strength, hydrothermal stability and structural integrity, swelling and water uptake properties of collagen scaffolds in the presence of gallic acid with EDC/NHS. The enzymatic attack of collagen scaffolds were also prevented upon treatment with gallic acid [14]. As Cr(III) is an inert and adopts stable configuration, it has been extensively used in leather industry [15,16]. However, its uptake in the chrome tanning process alone limits either the strength to achieve, colour or softness for the preparation of wide range of leather. To serve this purpose, different researchers have utilized vegetable tannins i.e. polyphenols along with chromium. Unfortunately, the uptake of either chromium or polyphenolic compounds
430
D. Jaikumar et al. / International Journal of Biological Macromolecules 96 (2017) 429–435
are also not complete resulting in huge amount of waste generation. Moreover, as chromium waste being highly toxic and environment pollutant, every industry looks for the containment of waste. In order to overcome this issue, we have synthesized and characterized a Cr(III) gallic acid complex and studied its biophysical properties with collagen. The rationale for this study using synthesized Cr(III)-polyphenol complex is to understand the binding behaviour of Cr(III) complex with the fibrous protein. The outcome of the work might help in understanding the uptake of Cr(III) during tanning process. In addition, by synthesizing the Cr(III) complex as single entity might reduce the multistep tanning procedure and also minimize the leaching of chromium. 2. Materials and methods 2.1. Materials Sodium acetate trihydrate, gallic acid and chromium(III) chloride was obtained from Sigma-Aldrich chemicals, Bangalore. All the buffers used in this study were filtered before carrying out any experiments. ESI mass spectrum of the complex was recorded with a Thermo-Finnigan LCQ-6000 Advantage Max ion trap mass spectrometer equipped with an electron spray source. The elemental composition of the synthesized compound was carried out using EURO EA 3000–Single CHNS analyser. 2.2. Extraction of collagen Acid soluble rat tail tendon (RTT) collagen type I was isolated according to the procedure described before [17]. Briefly, native rat tail tendons (RTT) were teased at 4 ◦ C and washed with 0.9% saline solution. After extensive salting out and extraction procedure, the collagen solution was dialyzed to remove any degraded product followed by centrifugation. The collagen concentration was determined from the hydroxyproline content as mentioned earlier. The purity of collagen was examined using gel electrophoresis and the molar concentration of collagen was determined by considering the average molecular weight of collagen as 300 kDa [18]. 2.3. Synthesis of chromium(III) gallate, [Cr(GA)2 ]− Chromium(III) gallate complex was prepared by mixing potassium chromate and gallic acid in equivalent molar ratio followed by incubation at 25 ◦ C for 24 h. The mixture was purified using a Sephadex G-25 column [19]. ESI–MS: m/z 413 [M2H2 O + Na+H]+ . C14 H12 CrNaO12 (446.23): calcd. C 37.64, H 2.7, Cr 11.63; found C 37.32, H 2.52, Cr 11.85. 2.4. CD studies The influence of synthesized Cr(gallate) and gallic acid on the conformation of collagen was studied using JASCO J-815 spectrometer under nitrogen atmosphere. The CD measurements were carried out using a quartz cell with light path of 0.1 cm at 25 ◦ C. The CD spectra were recorded in the far UV region (190–400 nm). An aqueous solution of collagen (0.5 M) in acetate buffer (pH 4.5, I = 20 × 10−2 M) were incubated with varying concentrations of [Cr(GA)2 ] and gallic acid (0.5–50 M) for 6 h at 25 ◦ C. 2.5. Viscosity measurements Viscometric experiments were carried out using Ostwald type viscometer of 5 mL capacity thermostated in a water bath at 25 ◦ C. The concentration of collagen (0.5 M) was kept fixed and the flow time measurements were carried out using varying amounts of gallic acid and [Cr(GA)2 ] (0–50 M). Here, the viscosity contribution
() due to collagen was measured as a function of the concentration of the complex. The flow time was measured using a manually operated timer at least three times and the average was taken. The viscosity was calculated using the relation, = (t − t0 )/t0 , where t0 is the flow time of buffer (pH 4.5, I = 20 × 10−2 M) and t is the flow time for each sample. The values of relative viscosity, /0 ( and 0 are the viscosity of collagen in the presence and absence of the additive) vs. ratio of [complex]/[collagen] were plotted [18]. 2.6. Hydrothermal shrinkage measurement The hydrothermal stability of the reconstituted collagen fibres treated with Cr(III) and gallic acid were determined by the standard method using a micro shrinkage tester [20]. The lyophilized treated and untreated collagen fibres in water were placed on the heating stage along with a microscope mounted over it. The rate of heating was maintained at 2 ◦ C/min. Hydrothermal shrinkage temperature was taken as the temperature at which the collagen fibre shrinks to one third of its original length. 2.7. Differential scanning calorimetry studies DSC thermograms of lyophilized treated/untreated collagen fibers were recorded in a micro differential scanning calorimeter Q-200 (TA instruments) at 1 ◦ C/min heating rate under nitrogen atmosphere. The temperature range used in the measurement was 20–90 ◦ C. The experiments were performed in duplicate. The collagen fibers were prepared by incubating the collagen with or without GA and [Cr(GA)2 ] in the ratio of 1:10 for 24 h followed by lyophilization. The apparent melting temperature was determined from the transition after baseline subtraction using empty pan. 2.8. FT-Infrared spectroscopic studies Fourier transform infrared spectroscopy (FT-IR) spectra for the treated and untreated collagen were obtained from the films after kept for drying in a desiccator for 24 h at room temperature by a JASCO 4700 FTIR spectrometer. All spectra were obtained with a resolution of 4 cm−1 in the range of 400–4000 cm−1 . The spectra plots represent the average of 32 scans. 2.9. Fibrillation assay The effect of [Cr(GA)2 ] and gallic acid on the fibril formation of collagen was assessed by initializing fibril formation at 25 ◦ C by mixing a final concentration of 0.5 M collagen with 50 mM phosphate buffer and 75 mM sodium chloride. The pH was adjusted to 7.5 using 0.5 M NaOH. The turbidity was measured at 400 nm using a Shimadzu UV-1700 spectrophotometer. The assay was performed by keeping collagen with [Cr(GA)2 ] and gallic acid in the ratio of 1:10. The rate of fibril formation was determined by calculating the time taken to reach half the value of the final turbidity (t1/2 ) [21]. 2.10. SEM imaging The collagen fibril structures after treatment with gallic acid and [Cr(GA)2 ] was analysed using SEM (PhenomTM Pro) with an accelerating voltage of 5 kV. The fibrils were formed as mentioned in fibrillation assay. Collagen(0.5 M) was treated with gallic acid or [Cr(GA)2 ] (5 M) in the molar ratio of 1:10 at pH 4.5 and adjusted to pH 7.5 by adding sodium hydroxide. These samples were loaded on the aluminium substrate, left dried and were thoroughly rinsed with distilled water before loading on to the metal stub.
D. Jaikumar et al. / International Journal of Biological Macromolecules 96 (2017) 429–435
431
Fig. 1. ESI mass spectrum of [Cr(GA)2 ].
2.11. AFM imaging To determine the packing of collagen before and after treatment with gallic acid and [Cr(GA)2 ], d-periodicity of the collagen were captured using NTEGRA PRIMA (NT-MDT, Russia) atomic force microscopy. The cantilever (CSG01 series) was used with a nominal spring constant of approx. 0.03N/m. A tip of 10 nm diameter was fixed at the free end of the cantilever. The sample consisting of collagen (0.5 M) and GA or [Cr(GA)2 ] (5 M) were loaded onto the mica substrate and kept for drying overnight. The instrument was used on contact mode with a scanning rate of 0.5 Hz. The d band measurements were calculated using the Nova PX software.
nism. The preparation of [Cr(GA)2 ] has been confirmed by ESI–MS in which the molecular ion was observed at m/z 413.25 which indicate that two gallic acid molecules coordinated to the chromium metal center with the loss of two water molecules under mass spectral condition (Fig. 1). An additional peak observed here at m/z 804.76 indicates the formation of dimeric species of the complex during ionization process in which one of the coordinated water form oxo species and shares with another chromium center. The concentration of chromium was estimated using alkali-peroxide method [22].
3.1. Circular dichroic studies 3. Results and discussion The present study discusses about the role of Cr(III) in gallic acid complex in the crosslinking of collagen. Although number of studies have been reported on polyphenols and flavonoid related molecules, the understanding of Cr(III) polyphenolic complex in collagen will provide vital information on the crosslinking mecha-
To determine the effect of [Cr(GA)2 ] and gallic acid induced crosslinks on the secondary structure of collagen, circular dichroic studies were performed by varying the concentrations of gallic acid (0–50 M) and [Cr(GA)2 ] (0–50 M) while keeping the concentration of collagen (0.5 M) constant. The triple helical content of the collagen was confirmed by monitoring a positive peak at 220 nm with a crossover point at 210 nm and a large negative peak
432
D. Jaikumar et al. / International Journal of Biological Macromolecules 96 (2017) 429–435
Fig. 2. CD spectra of collagen (0.5 M) in the absence and presence of gallic acid (0–50 M).
Fig. 4. FT-IR spectra of collagen film before and after treatment with GA and [Cr(GA)2 ].
alterations takes place in collagen upon treatment with [Cr(GA)2 ] and in consistent with results of gallic acid. 3.2. FT-IR studies of [Cr(GA)2 ] treated and untreated collagen
Fig. 3. CD spectra of collagen (0.5 M) in the absence and presence of [Cr(GA)2 ] (0–50 M). Table 1 Rpn ratio of collagen in the absence and presence of gallic acid and [Cr(GA)2 ]. Samples
RPN
Collagen (0.5 M) +Gallic acid (50 M) +[Cr(GA)2 ] (50 M)
0.133 0.136 0.140
at 197 nm [23]. The CD spectra of collagen with increasing concentrations of [Cr(GA)2 ] retained the structural conformation of collagen even after forming crosslink. As seen from Figs. 2 and 3, however, only marginal variation in amplitude was observed for collagen treated with either [Cr(GA)2 ] or gallic acid. The parameter Rpn denotes the ratio of positive peak intensity to the negative peak intensity and establishes the triple helical information of collagen [24]. As seen from Table 1, the Rpn ratio of collagen was determined as 0.13 and with increasing concentrations of [Cr(GA)2 ] treated collagen or gallic acid, only minor deviations (0.14) from the native was observed. Generally, a red shift of negative peak and disappearance of positive peak would be observed for a denaturation process [25,26]. Since no such variations was observed here indicate that the small changes in the CD spectra of Cr(III) treated collagen were not due to denaturation or loss of triple helicity. From the circular dichroic results, it is evident that no major structural
To assess the backbone conformational change of collagen before and after treatment with chromium(III) complex, FTIR spectral studies were performed. The triple helical nature of collagen exhibits characteristic amide A and B bands in IR spectra with N H stretching vibration appeared at 3270 cm−1 and 3070 cm−1 , respectively. In addition, the stretching frequency of carbonyl group( C O ) appears at 1642 cm−1 was observed (Fig. 4). From the IR data, no shift in the stretching frequency of amide bonds in collagen was observed upon addition of [Cr(GA)2 ] as well as with GA compared to native collagen. The absence of spectral shift observed here indicate that the coordinated water molecule in [Cr(GA)2 ] might form hydrogen bonding with amide backbone along with GA moiety. The results observed here is similar to previously reported polyphenol such as procyanidin in which even increasing concentrations of procyanidin did not alter backbone structure of collagen [27] even though it involves in hydrogen bonding. 3.3. Viscosity studies Studies on the viscosity of collagen were carried out to understand the interaction and influence of the additive on collagen. Relative viscosity of collagen was measured in the presence of varying concentrations of the gallic acid and [Cr(GA)2 ]. A plot of relative viscosity (/0 ) against 1/R (R = [collagen]/[complex]) is shown in Fig. 5. In the case of gallic acid treated collagen, a slight increase in relative viscosity was observed upon addition of increasing amounts of gallic acid. On the other hand, relative viscosity significantly decreased with increasing concentrations of [Cr(GA)2 ]. The viscosity of Cr(III) complex treated collagen decreased from 1.06 to 0.17 whereas it remained constant at 1.02 for gallic acid treated collagen. Even though [Cr(GA)2 ] can bring about the long range ordering of the collagenous matrix, it did not lead to any aggregation of the protein. Moreover, the observed significant decrease in viscosity could be due to the presence of two aquo molecules in Cr(III) complex forms hydrogen bonding with collagen backbone in addition to GA moiety and enhance the crosslink formation thereby leading to decrease in the hydrodynamic volume of the collagen molecule [18]. From the viscosity results, it is clearly revealed that [Cr(GA)2 ] promotes the crosslinking in collagen significantly compared to gallic acid.
D. Jaikumar et al. / International Journal of Biological Macromolecules 96 (2017) 429–435
433
Fig. 5. Relative viscosity plot of collagen (0.5 M) in the absence and presence of [Cr(GA)2 ] and gallic acid(0–50 M). Table 2 Hydrothermal shrinkage temperature of collagen treated with gallic acid and [Cr(GA)2 ]. Samples
Hydrothermal shrinkage temperature
Native collagen +Gallic acid +[Cr(GA)2 ]
52 ± 1 ◦ C 53 ± 1 ◦ C 58 ± 1 ◦ C
Fig. 6. DSC thermograms of collagen film before and after treatment with [Cr(GA)2 ].
3.4. Thermal stability studies To determine the overall stability of collagenous matrix due to its long range ordering, shrinkage temperature was measured before and after treatment with [Cr(GA)2 ] and compared with collagen treated with gallic acid. As collagen fibres are held together by hydrogen bonds between the adjacent chains of their polypeptide helical strands, upon heating, these bonds break and leads to structural modifications. Moreover, the hydrothermal contraction of collagen occurs due to their internal molecular rearrangement and results in reduction in the length of the collagen fiber. The shrinkage temperature of native, gallic acid and [Cr(GA)2 ] treated fibers were determined as 52, 53 and 58 ± 1 ◦ C, respectively (Table 2). From the shrinkage temperature, gallic acid treated collagen fibers showed negligible increase compared to that of native. However, an increase of 6 ◦ C with [Cr(GA)2 ] treated collagen revealed that the introduction of Cr(III) in the gallic acid complex enhances the stability of the collagen structure compared to gallic acid alone [28]. Similarly, the calorimetric studies also showed an increase of 7 ◦ C from 66 to 73 ◦ C for [Cr(GA)2 ] treated collagen compared to native collagen (Fig. 6). The increase in denaturation temperature might be due to crosslinking of Cr(III) complex with collagen triple helical backbone by forming hydrogen bonds between coordinated water molecules in Cr(GA)2 complex and amide backbone in collagen. The increase in denaturation temperature of collagen after treatment with chromium complex shows that [Cr(GA)2 ] provides improved stability and mechanical strength of the collagen. 3.5. Fibrillation kinetics In order to study the extent of crosslinking, fibrillation kinetics was performed on collagen in the absence and presence of gallic acid, CrCl3 and [Cr(GA)2 ]. As fibrillogenesis involves two-step process, i.e., aggregation of individual helical molecules into nuclei and the growth of nuclei into fibrils, the rate of fibril formation was measured by its turbidity [29]. Generally, crosslinking efficacy was determined by monitoring the increase in turbidity at 400 nm and also the turbidity curve (a lag, growth and a plateau
Fig. 7. Fibrillation kinetics of collagen (0.5 M) in the absence and presence of [Cr(GA)2 ] and gallic acid(5 M). Table 3 Fibrillation kinetics of collagen untreated/treated with gallic acid and [Cr(GA)2 ]. samples
h(final turbidity)
t(1/2) (min)
Native collagen Gallic acid Cr(III)-gallate
0.139 0.128 0.162
6.59 5.67 15.34
phase). While the growth phase exhibits a rapid change in its turbidity, the thickness of the stable fibrils formed is measured by the absorbance levels at the plateau phase. The higher absorbance at 400 nm reflects the increased thickness of the fibrils. The effect of any small molecules on the fibril formation will be determined by its final turbidity (h) and the time taken to reach half the value of its turbidity (t1/2 ). From the turbidity measurements, the lag phase for the native or gallic acid treated collagen was almost similar while a significant delay of nine minutes in fibrillation process was observed for [Cr(GA)2 ] (Fig. 7 and Table 3). In the case of gallic acid, though polyphenolic groups tend to delay the fibril formation, in the present case, it was not observed. However, by forming complex with chromium, the inhibition in fibril formation was significant and can be explained in terms of synergistic effect of Cr(III) and gallic acid resulting in strong crosslinking. In addition, two of the water molecules in [Cr(GA)2 ] might involve in hydrogen
434
D. Jaikumar et al. / International Journal of Biological Macromolecules 96 (2017) 429–435
Fig. 8. Scanning electron micrographs of (a) native collagen(0.5 M) (b) with gallic acid(5 M) and (c) with [Cr(GA)2 ] (5 M) (Magnification: 5300×; ratio of [Collagen]: GA or [Cr(GA)2 ] = 1:10).
Fig. 9. Atomic force microscopic images of collagen of (a) native (0.5 M) (b) treated with gallic acid (5 M)and (c) treated with [Cr(GA)2 ] (5 M) (ratio of [Collagen]: GA/[Cr(GA)2 ] = 1:10).
bonding and provides additional stability onto the fibrous matrix of collagen. 3.6. SEM analysis The morphological analysis of collagen fibril before and after treatment with [Cr(GA)2 ] were carried out using scanning electron microscope. It was found that the [Cr(GA)2 ] complex significantly influence collagen fibril assembly and its diameter. As seen from Fig. 8, collagen in its native form and on treatment with gallic acid forms thickened fibres whereas [Cr(GA)2 ] treated collagen exhibited thinly spread collagenous fibrous network. The thickness of the fibres were measured and found to be 10 m and 11 m for native and gallic acid treated collagen fibres, respectively. However, collagen treated with [Cr(GA)2 ]+ displayed the microfibril formation with thickness in nanometer level. The morphological change observed with respect to [Cr(GA)2 ] further supports the efficient crosslinking process by inhibiting the fibril formation compared to native and gallic acid treated collagen. 3.7. AFM imaging The changes in the secondary structure of collagen brought about by the metal complexes has been investigated by AFM imaging. AFM provides evidence for the self-assembly of collagen. The topological distribution and the interaction of metal ions onto the collagenous matrix can be explained using AFM images. AFM image of Type I collagen has many grooves and ridges in which collagen monomers are staggered into a quarterly fibrillar structure with a d-periodicity of 64 nm. The d-periodicity values observed for native
collagen, gallic acid and [Cr(GA)2 ] treated collagenous fibres are 62, 68.9 and 58.9 nm, respectively (Fig. 9). Gayathri et al., have shown a variation in d-periodicity of nearly 8 nm when collagen treated with various species of Cr(III) complexes compared to native collagen [30]. The observed marginal difference in the d-periodicity reveal that [Cr(GA)2 ]+ complex might displace the water involved in hydration network of the collagen, thereby resulting in structural rearrangement of collagen [30]. The slight variations in the d-period values indicate that no major structural alterations occurred. Similarly, the less difference in the d period values of gallic acid and [Cr(GA)2 ] treated collagen does not cause structural interruptions in the matrix. Slight loosening of triple helical configuration might have resulted in a decreased d period value of 58.9 nm. Also, the difference in d periodicity might be due to the displacement of water involved in the hydration network of collagen caused by the metal ions thereby affecting its intra and inter chemical bonds compared to gallic acid. 4. Conclusion Altogether, the data from the interaction of [Cr(GA)2 ] with collagen reveals that (i) the presence of Cr in the form of chromium complex did not alter the triple helical nature of the collagen; (ii) with [Cr(GA)2 ], the hydrothermal stability of the collagen improved by 6 ◦ C compared to gallic acid; (iii) the viscosity of collagen significantly reduced in the presence of complex; (iv) a significant delay in fibril formation by the complex; (v) SEM analysis confirms the inhibition of fibril formation in the presence of [Cr(GA)2 ] and (vi) reduction in d-periodicity of collagen with [Cr(GA)2 ]. The results from this study clearly provides the information on the presence
D. Jaikumar et al. / International Journal of Biological Macromolecules 96 (2017) 429–435
of Cr(III) in the form of polyphenolic complex i.e. [Cr(GA)2 ] further stabilizes collagen structure as well as crosslinking and might help in development of new products for leather tanning. Acknowledgments This research is supported by Science and Technology Revolution in Leather with a Green Touch (STRAIT) Institutional Project. V.G.V thanks DST-SERB for Ramanujan fellowship (SR/S2/RJN07/2011). The authors thank the reviewers for their valuable suggestions. References [1] S. Ricard-Blum, The collagen family, Cold Spring Harb. Perspect. Biol. 3 (1) (2011) a004978. [2] J. Kastelic, E. Baer, J.F.V Vincent, J.D. Currey (Eds.), Cambridge University Press, 397–435 (1980). [3] N.S. Demina, S.V. Lysenko, Microbiology 65 (1996) 257–265. [4] D.J. Harrington, Infect. Immunol. 64 (1996) 1885–1891. [5] J.R. Rao, R. Gayatri, R. Rajaram, B.U. Nair, T. Ramasami, Chromium(III) hydrolytic oligomers: their relevance to protein binding, Biochem. Biophys. Acta 1472 (3) (1999) 595–602. [6] H.Y. Shrivastava, B.U. Nair, J. Biomol. Struct. Dyn. 20 (2003) 575. [7] S. Ahmed, Arthritis Res. Ther. 12 (2010) 1–9. [8] A.A. Haroun, S.A. Toumy, J. Appl. Polym. Sci. 116 (2010) 770–776. [9] E. Haslam, Phytochemistry 68 (2007) 2713–2721. [10] B. Madhan, G. Krishnamoorthy, J.R. Rao, B.U. Nair, Role of green tea polyphenols in the inhibition of collagenolytic activity by collagenase, Int. J. Biol. Macromol. 41 (1) (2007) 16–22.
435
[11] J.C. Isenberg, D.T. Simionescu, N.R. Vyavahare, Biomaterials 26 (2005) 1237–1245. [12] M.E. Tedder, J. Liao, B. Weed, C. Stabler, H. Zhang, A. Simionescu, D.T. Simionescu, Tissue Eng. Part A 159 (2009) 1257–1268. [13] G. Krishnamoorthy, B. Madhan, S. Sadulla, J.R. Rao, M. Madhulatha, J. Appl. Polym. Sci. 108 (2008) 199–205. [14] G. Krishnamoorthy, R. Selvakumar, T.P. Sastry, S. Sadulla, A.B. Mandal, M. Doble, Mater. Sci. Eng. C 42 (2014) 164–171. [15] J.R. Rao, R. Gayatri, R. Rajaram, B.U. Nair, T. Ramasami, Biochem. Biophys. Acta 1472 (1999) 595–602. [16] E. Kong, S.J. Herzog, Inorg. Nucl. Chem. 32 (1970) 585–599. [17] G. Chandrakasan, D.A. Torchia, K.A. Piez, Preparation of intact monomeric collagen from rat tail tendon and skin and the structure of the nonhelical ends in solution, J. Biol. Chem. 251 (1976) 6062–6067. [18] N.N. Fathima, J.R. Rao, B.U. Nair, J. Inorg. Biochem. 100 (2006) 1774–1780. [19] H. Arakawa, M.-S. Tang, Chem. Res. Toxicol. 2 (2008) 1284–1289. [20] R. Borasky, G.C. Nutting, J. Am. Leather Chem. Assoc. 44 (1949) 830–841. [21] K.V. Srivatsan, R. Lakra, K. Purna Sai, M.S. Kiran, J. Mater. Chem. B 4 (2016) 1437–1447. [22] J.J. Lingane, R.L. Pecsok, Preparation of standard chromous sulfate or chromous chloride solutions of determinate concentration, Anal. Chem. 20 (5) (1948) 425–428. [23] M.L. Tiffany, S. Krim, Biopolymers 8 (1969) 347–359. [24] N. Greenfield, Nat. Protoc. 1 (2007) 2876–2890. [25] N.N. Fathima, A. Dhathathreyan, Int. J. Biol. Macromol. 45 (2009) 274–278. [26] Y. Li, Y. Li, Z. Du, G. Li, Thermochim. Acta 469 (2008) 71–76. [27] L. He, C. Mu, J. Shi, Q. Zhang, B. Shi, W. Lin, Modification of collagen with a natural cross-linker, procyanidin, Int. J. Biol. Macromol. 48 (2) (2011) 354–359. [28] R. Gayatri, R. Rajaram, B.U. Nair, F. Chandrasekaran, T. Ramasami, Proc. Indian Acad. Sci. 111 (1999) 133–145. [29] K.E. Kadler, D.F. Holmes, J.A. Trotter, J.A. Chapman, Biochem. J. 316 (1996) 1–11. [30] R. Gayatri, A.K. Sharma, R. Rajaram, T. Ramasami, Biochem. Biophys. Res. Commun. 283 (2001) 229–235.
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Effect of chromium(III) gallate complex on stabilization of collagen Dhanya Jaikumar, Babu Baskaran, V.G. Vaidyanathan ∗ Advanced Materials Laboratory and Academy of Scientific and Innovative Research (AcSIR), CSIR-Central Leather Research Institute, Adyar, Chennai 600 020, India
a r t i c l e
i n f o
Article history: Received 6 October 2016 Received in revised form 16 December 2016 Accepted 17 December 2016 Available online 21 December 2016 Keywords: Collagen Chromium (III) Crosslinking Fibrils Viscosity Polyphenols
a b s t r a c t To improve the stability of the collagen, here we studied the interaction of chromium(III) polyphenolic complex, [Cr(GA)2 ] (GA: Gallic acid) with collagen using various spectroscopic techniques. Circular dichroism studies show that the [Cr(GA)2 ] and gallic acid did not induce any structural perturbations on the triple helix of the collagen. Both differential scanning calorimetric(DSC) data and micro-shrinkage temperature studies showed that [Cr(GA)2 ] stabilized the collagen by 6 ± 1 ◦ C compared to gallic acid. Hydrodynamic studies revealed that the viscosity of collagen drastically reduced in the presence of [Cr(GA)2 ] while gallic acid did not. Fibrillation assay displayed a significant delay in fibril formation with Cr(III) complex compared to gallic acid treated collagen. The inhibition of fibril formation was further confirmed by microscopic data in which collagen fibres are seen with GA while [Cr(GA)2 ] treated collagen exhibit a thin microfibrils. From AFM studies, the d-periodicity of collagen was found to be decreased with [Cr(GA)2 ] while increased with gallic acid. The present study deliberate the advantage of metal complex containing polyphenolic ligand as crosslinking agent due to its synergistic effect of both metal center as well as polyphenolic groups in the stabilization of collagen structure. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Collagen is an essential structural protein as well an important component of extracellular matrix. It accounts for 30% of the total protein content of the body helping in cell adhesion, its proliferation, migration, etc. There are 28 fibrillar and non-fibrillar types of collagen identified to date [1]. Of which, usually the structure of Type I collagen, its hierarchical ordering and self-assembly processes are highlighted. Each alpha chain is a left-handed polyproline intertwined to form a right-handed triple helix. The triple helical structure of collagen composed of three individual alpha chains with Gly-X-Y as repeated sequence observed throughout the length of the chain [2]. Moreover, structure is stabilized by hydrogen bonds with intra- and intermolecular forces. Due to its biodegradability and poor mechanical stability, collagenase attacks the native collagen at physiological pH, temperature and ionic strength easily [3,4]. To improve the mechanical stability and minimize the biodegradation rate, crosslinking of collagen is an essential step. A number of studies have been reported on stabilization of type I collagen by small molecules [5]. Specifically, transition
∗ Corresponding author at: Advanced Materials Laboratory, CSIR-Central Leather Research Institute, Adyar, Chennai 600020, India. E-mail address: [email protected] (V.G. Vaidyanathan). http://dx.doi.org/10.1016/j.ijbiomac.2016.12.049 0141-8130/© 2016 Elsevier B.V. All rights reserved.
metal based complexes are known to bind with collagen and enhance the stability. In particular, chromium with an oxidation state of +3 is preferred over +6 due to its lesser carcinogenicity and binds with aspartic and glutamic residues of collagen by aqua ligand substitution [6]. Similarly, studies on the interaction of polyphenolic compounds with proteins have also been reported for its better stability [7–9]. In addition, it has diverse applications in the pharmaceutical industries. Epigallocatechingallate (EGCG), catechin and gallic acid etc., has been reported to form strong complexes with the proteins [10]. As polyphenolic compounds possess aromatic, hydroxyl and acid groups, it can bind to the proteins through four key interactions i.e., non-covalent, covalent, ionic and H-bonds [11–13]. Krishnamoorthy et al., demonstrated the increased mechanical strength, hydrothermal stability and structural integrity, swelling and water uptake properties of collagen scaffolds in the presence of gallic acid with EDC/NHS. The enzymatic attack of collagen scaffolds were also prevented upon treatment with gallic acid [14]. As Cr(III) is an inert and adopts stable configuration, it has been extensively used in leather industry [15,16]. However, its uptake in the chrome tanning process alone limits either the strength to achieve, colour or softness for the preparation of wide range of leather. To serve this purpose, different researchers have utilized vegetable tannins i.e. polyphenols along with chromium. Unfortunately, the uptake of either chromium or polyphenolic compounds
430
D. Jaikumar et al. / International Journal of Biological Macromolecules 96 (2017) 429–435
are also not complete resulting in huge amount of waste generation. Moreover, as chromium waste being highly toxic and environment pollutant, every industry looks for the containment of waste. In order to overcome this issue, we have synthesized and characterized a Cr(III) gallic acid complex and studied its biophysical properties with collagen. The rationale for this study using synthesized Cr(III)-polyphenol complex is to understand the binding behaviour of Cr(III) complex with the fibrous protein. The outcome of the work might help in understanding the uptake of Cr(III) during tanning process. In addition, by synthesizing the Cr(III) complex as single entity might reduce the multistep tanning procedure and also minimize the leaching of chromium. 2. Materials and methods 2.1. Materials Sodium acetate trihydrate, gallic acid and chromium(III) chloride was obtained from Sigma-Aldrich chemicals, Bangalore. All the buffers used in this study were filtered before carrying out any experiments. ESI mass spectrum of the complex was recorded with a Thermo-Finnigan LCQ-6000 Advantage Max ion trap mass spectrometer equipped with an electron spray source. The elemental composition of the synthesized compound was carried out using EURO EA 3000–Single CHNS analyser. 2.2. Extraction of collagen Acid soluble rat tail tendon (RTT) collagen type I was isolated according to the procedure described before [17]. Briefly, native rat tail tendons (RTT) were teased at 4 ◦ C and washed with 0.9% saline solution. After extensive salting out and extraction procedure, the collagen solution was dialyzed to remove any degraded product followed by centrifugation. The collagen concentration was determined from the hydroxyproline content as mentioned earlier. The purity of collagen was examined using gel electrophoresis and the molar concentration of collagen was determined by considering the average molecular weight of collagen as 300 kDa [18]. 2.3. Synthesis of chromium(III) gallate, [Cr(GA)2 ]− Chromium(III) gallate complex was prepared by mixing potassium chromate and gallic acid in equivalent molar ratio followed by incubation at 25 ◦ C for 24 h. The mixture was purified using a Sephadex G-25 column [19]. ESI–MS: m/z 413 [M2H2 O + Na+H]+ . C14 H12 CrNaO12 (446.23): calcd. C 37.64, H 2.7, Cr 11.63; found C 37.32, H 2.52, Cr 11.85. 2.4. CD studies The influence of synthesized Cr(gallate) and gallic acid on the conformation of collagen was studied using JASCO J-815 spectrometer under nitrogen atmosphere. The CD measurements were carried out using a quartz cell with light path of 0.1 cm at 25 ◦ C. The CD spectra were recorded in the far UV region (190–400 nm). An aqueous solution of collagen (0.5 M) in acetate buffer (pH 4.5, I = 20 × 10−2 M) were incubated with varying concentrations of [Cr(GA)2 ] and gallic acid (0.5–50 M) for 6 h at 25 ◦ C. 2.5. Viscosity measurements Viscometric experiments were carried out using Ostwald type viscometer of 5 mL capacity thermostated in a water bath at 25 ◦ C. The concentration of collagen (0.5 M) was kept fixed and the flow time measurements were carried out using varying amounts of gallic acid and [Cr(GA)2 ] (0–50 M). Here, the viscosity contribution
() due to collagen was measured as a function of the concentration of the complex. The flow time was measured using a manually operated timer at least three times and the average was taken. The viscosity was calculated using the relation, = (t − t0 )/t0 , where t0 is the flow time of buffer (pH 4.5, I = 20 × 10−2 M) and t is the flow time for each sample. The values of relative viscosity, /0 ( and 0 are the viscosity of collagen in the presence and absence of the additive) vs. ratio of [complex]/[collagen] were plotted [18]. 2.6. Hydrothermal shrinkage measurement The hydrothermal stability of the reconstituted collagen fibres treated with Cr(III) and gallic acid were determined by the standard method using a micro shrinkage tester [20]. The lyophilized treated and untreated collagen fibres in water were placed on the heating stage along with a microscope mounted over it. The rate of heating was maintained at 2 ◦ C/min. Hydrothermal shrinkage temperature was taken as the temperature at which the collagen fibre shrinks to one third of its original length. 2.7. Differential scanning calorimetry studies DSC thermograms of lyophilized treated/untreated collagen fibers were recorded in a micro differential scanning calorimeter Q-200 (TA instruments) at 1 ◦ C/min heating rate under nitrogen atmosphere. The temperature range used in the measurement was 20–90 ◦ C. The experiments were performed in duplicate. The collagen fibers were prepared by incubating the collagen with or without GA and [Cr(GA)2 ] in the ratio of 1:10 for 24 h followed by lyophilization. The apparent melting temperature was determined from the transition after baseline subtraction using empty pan. 2.8. FT-Infrared spectroscopic studies Fourier transform infrared spectroscopy (FT-IR) spectra for the treated and untreated collagen were obtained from the films after kept for drying in a desiccator for 24 h at room temperature by a JASCO 4700 FTIR spectrometer. All spectra were obtained with a resolution of 4 cm−1 in the range of 400–4000 cm−1 . The spectra plots represent the average of 32 scans. 2.9. Fibrillation assay The effect of [Cr(GA)2 ] and gallic acid on the fibril formation of collagen was assessed by initializing fibril formation at 25 ◦ C by mixing a final concentration of 0.5 M collagen with 50 mM phosphate buffer and 75 mM sodium chloride. The pH was adjusted to 7.5 using 0.5 M NaOH. The turbidity was measured at 400 nm using a Shimadzu UV-1700 spectrophotometer. The assay was performed by keeping collagen with [Cr(GA)2 ] and gallic acid in the ratio of 1:10. The rate of fibril formation was determined by calculating the time taken to reach half the value of the final turbidity (t1/2 ) [21]. 2.10. SEM imaging The collagen fibril structures after treatment with gallic acid and [Cr(GA)2 ] was analysed using SEM (PhenomTM Pro) with an accelerating voltage of 5 kV. The fibrils were formed as mentioned in fibrillation assay. Collagen(0.5 M) was treated with gallic acid or [Cr(GA)2 ] (5 M) in the molar ratio of 1:10 at pH 4.5 and adjusted to pH 7.5 by adding sodium hydroxide. These samples were loaded on the aluminium substrate, left dried and were thoroughly rinsed with distilled water before loading on to the metal stub.
D. Jaikumar et al. / International Journal of Biological Macromolecules 96 (2017) 429–435
431
Fig. 1. ESI mass spectrum of [Cr(GA)2 ].
2.11. AFM imaging To determine the packing of collagen before and after treatment with gallic acid and [Cr(GA)2 ], d-periodicity of the collagen were captured using NTEGRA PRIMA (NT-MDT, Russia) atomic force microscopy. The cantilever (CSG01 series) was used with a nominal spring constant of approx. 0.03N/m. A tip of 10 nm diameter was fixed at the free end of the cantilever. The sample consisting of collagen (0.5 M) and GA or [Cr(GA)2 ] (5 M) were loaded onto the mica substrate and kept for drying overnight. The instrument was used on contact mode with a scanning rate of 0.5 Hz. The d band measurements were calculated using the Nova PX software.
nism. The preparation of [Cr(GA)2 ] has been confirmed by ESI–MS in which the molecular ion was observed at m/z 413.25 which indicate that two gallic acid molecules coordinated to the chromium metal center with the loss of two water molecules under mass spectral condition (Fig. 1). An additional peak observed here at m/z 804.76 indicates the formation of dimeric species of the complex during ionization process in which one of the coordinated water form oxo species and shares with another chromium center. The concentration of chromium was estimated using alkali-peroxide method [22].
3.1. Circular dichroic studies 3. Results and discussion The present study discusses about the role of Cr(III) in gallic acid complex in the crosslinking of collagen. Although number of studies have been reported on polyphenols and flavonoid related molecules, the understanding of Cr(III) polyphenolic complex in collagen will provide vital information on the crosslinking mecha-
To determine the effect of [Cr(GA)2 ] and gallic acid induced crosslinks on the secondary structure of collagen, circular dichroic studies were performed by varying the concentrations of gallic acid (0–50 M) and [Cr(GA)2 ] (0–50 M) while keeping the concentration of collagen (0.5 M) constant. The triple helical content of the collagen was confirmed by monitoring a positive peak at 220 nm with a crossover point at 210 nm and a large negative peak
432
D. Jaikumar et al. / International Journal of Biological Macromolecules 96 (2017) 429–435
Fig. 2. CD spectra of collagen (0.5 M) in the absence and presence of gallic acid (0–50 M).
Fig. 4. FT-IR spectra of collagen film before and after treatment with GA and [Cr(GA)2 ].
alterations takes place in collagen upon treatment with [Cr(GA)2 ] and in consistent with results of gallic acid. 3.2. FT-IR studies of [Cr(GA)2 ] treated and untreated collagen
Fig. 3. CD spectra of collagen (0.5 M) in the absence and presence of [Cr(GA)2 ] (0–50 M). Table 1 Rpn ratio of collagen in the absence and presence of gallic acid and [Cr(GA)2 ]. Samples
RPN
Collagen (0.5 M) +Gallic acid (50 M) +[Cr(GA)2 ] (50 M)
0.133 0.136 0.140
at 197 nm [23]. The CD spectra of collagen with increasing concentrations of [Cr(GA)2 ] retained the structural conformation of collagen even after forming crosslink. As seen from Figs. 2 and 3, however, only marginal variation in amplitude was observed for collagen treated with either [Cr(GA)2 ] or gallic acid. The parameter Rpn denotes the ratio of positive peak intensity to the negative peak intensity and establishes the triple helical information of collagen [24]. As seen from Table 1, the Rpn ratio of collagen was determined as 0.13 and with increasing concentrations of [Cr(GA)2 ] treated collagen or gallic acid, only minor deviations (0.14) from the native was observed. Generally, a red shift of negative peak and disappearance of positive peak would be observed for a denaturation process [25,26]. Since no such variations was observed here indicate that the small changes in the CD spectra of Cr(III) treated collagen were not due to denaturation or loss of triple helicity. From the circular dichroic results, it is evident that no major structural
To assess the backbone conformational change of collagen before and after treatment with chromium(III) complex, FTIR spectral studies were performed. The triple helical nature of collagen exhibits characteristic amide A and B bands in IR spectra with N H stretching vibration appeared at 3270 cm−1 and 3070 cm−1 , respectively. In addition, the stretching frequency of carbonyl group( C O ) appears at 1642 cm−1 was observed (Fig. 4). From the IR data, no shift in the stretching frequency of amide bonds in collagen was observed upon addition of [Cr(GA)2 ] as well as with GA compared to native collagen. The absence of spectral shift observed here indicate that the coordinated water molecule in [Cr(GA)2 ] might form hydrogen bonding with amide backbone along with GA moiety. The results observed here is similar to previously reported polyphenol such as procyanidin in which even increasing concentrations of procyanidin did not alter backbone structure of collagen [27] even though it involves in hydrogen bonding. 3.3. Viscosity studies Studies on the viscosity of collagen were carried out to understand the interaction and influence of the additive on collagen. Relative viscosity of collagen was measured in the presence of varying concentrations of the gallic acid and [Cr(GA)2 ]. A plot of relative viscosity (/0 ) against 1/R (R = [collagen]/[complex]) is shown in Fig. 5. In the case of gallic acid treated collagen, a slight increase in relative viscosity was observed upon addition of increasing amounts of gallic acid. On the other hand, relative viscosity significantly decreased with increasing concentrations of [Cr(GA)2 ]. The viscosity of Cr(III) complex treated collagen decreased from 1.06 to 0.17 whereas it remained constant at 1.02 for gallic acid treated collagen. Even though [Cr(GA)2 ] can bring about the long range ordering of the collagenous matrix, it did not lead to any aggregation of the protein. Moreover, the observed significant decrease in viscosity could be due to the presence of two aquo molecules in Cr(III) complex forms hydrogen bonding with collagen backbone in addition to GA moiety and enhance the crosslink formation thereby leading to decrease in the hydrodynamic volume of the collagen molecule [18]. From the viscosity results, it is clearly revealed that [Cr(GA)2 ] promotes the crosslinking in collagen significantly compared to gallic acid.
D. Jaikumar et al. / International Journal of Biological Macromolecules 96 (2017) 429–435
433
Fig. 5. Relative viscosity plot of collagen (0.5 M) in the absence and presence of [Cr(GA)2 ] and gallic acid(0–50 M). Table 2 Hydrothermal shrinkage temperature of collagen treated with gallic acid and [Cr(GA)2 ]. Samples
Hydrothermal shrinkage temperature
Native collagen +Gallic acid +[Cr(GA)2 ]
52 ± 1 ◦ C 53 ± 1 ◦ C 58 ± 1 ◦ C
Fig. 6. DSC thermograms of collagen film before and after treatment with [Cr(GA)2 ].
3.4. Thermal stability studies To determine the overall stability of collagenous matrix due to its long range ordering, shrinkage temperature was measured before and after treatment with [Cr(GA)2 ] and compared with collagen treated with gallic acid. As collagen fibres are held together by hydrogen bonds between the adjacent chains of their polypeptide helical strands, upon heating, these bonds break and leads to structural modifications. Moreover, the hydrothermal contraction of collagen occurs due to their internal molecular rearrangement and results in reduction in the length of the collagen fiber. The shrinkage temperature of native, gallic acid and [Cr(GA)2 ] treated fibers were determined as 52, 53 and 58 ± 1 ◦ C, respectively (Table 2). From the shrinkage temperature, gallic acid treated collagen fibers showed negligible increase compared to that of native. However, an increase of 6 ◦ C with [Cr(GA)2 ] treated collagen revealed that the introduction of Cr(III) in the gallic acid complex enhances the stability of the collagen structure compared to gallic acid alone [28]. Similarly, the calorimetric studies also showed an increase of 7 ◦ C from 66 to 73 ◦ C for [Cr(GA)2 ] treated collagen compared to native collagen (Fig. 6). The increase in denaturation temperature might be due to crosslinking of Cr(III) complex with collagen triple helical backbone by forming hydrogen bonds between coordinated water molecules in Cr(GA)2 complex and amide backbone in collagen. The increase in denaturation temperature of collagen after treatment with chromium complex shows that [Cr(GA)2 ] provides improved stability and mechanical strength of the collagen. 3.5. Fibrillation kinetics In order to study the extent of crosslinking, fibrillation kinetics was performed on collagen in the absence and presence of gallic acid, CrCl3 and [Cr(GA)2 ]. As fibrillogenesis involves two-step process, i.e., aggregation of individual helical molecules into nuclei and the growth of nuclei into fibrils, the rate of fibril formation was measured by its turbidity [29]. Generally, crosslinking efficacy was determined by monitoring the increase in turbidity at 400 nm and also the turbidity curve (a lag, growth and a plateau
Fig. 7. Fibrillation kinetics of collagen (0.5 M) in the absence and presence of [Cr(GA)2 ] and gallic acid(5 M). Table 3 Fibrillation kinetics of collagen untreated/treated with gallic acid and [Cr(GA)2 ]. samples
h(final turbidity)
t(1/2) (min)
Native collagen Gallic acid Cr(III)-gallate
0.139 0.128 0.162
6.59 5.67 15.34
phase). While the growth phase exhibits a rapid change in its turbidity, the thickness of the stable fibrils formed is measured by the absorbance levels at the plateau phase. The higher absorbance at 400 nm reflects the increased thickness of the fibrils. The effect of any small molecules on the fibril formation will be determined by its final turbidity (h) and the time taken to reach half the value of its turbidity (t1/2 ). From the turbidity measurements, the lag phase for the native or gallic acid treated collagen was almost similar while a significant delay of nine minutes in fibrillation process was observed for [Cr(GA)2 ] (Fig. 7 and Table 3). In the case of gallic acid, though polyphenolic groups tend to delay the fibril formation, in the present case, it was not observed. However, by forming complex with chromium, the inhibition in fibril formation was significant and can be explained in terms of synergistic effect of Cr(III) and gallic acid resulting in strong crosslinking. In addition, two of the water molecules in [Cr(GA)2 ] might involve in hydrogen
434
D. Jaikumar et al. / International Journal of Biological Macromolecules 96 (2017) 429–435
Fig. 8. Scanning electron micrographs of (a) native collagen(0.5 M) (b) with gallic acid(5 M) and (c) with [Cr(GA)2 ] (5 M) (Magnification: 5300×; ratio of [Collagen]: GA or [Cr(GA)2 ] = 1:10).
Fig. 9. Atomic force microscopic images of collagen of (a) native (0.5 M) (b) treated with gallic acid (5 M)and (c) treated with [Cr(GA)2 ] (5 M) (ratio of [Collagen]: GA/[Cr(GA)2 ] = 1:10).
bonding and provides additional stability onto the fibrous matrix of collagen. 3.6. SEM analysis The morphological analysis of collagen fibril before and after treatment with [Cr(GA)2 ] were carried out using scanning electron microscope. It was found that the [Cr(GA)2 ] complex significantly influence collagen fibril assembly and its diameter. As seen from Fig. 8, collagen in its native form and on treatment with gallic acid forms thickened fibres whereas [Cr(GA)2 ] treated collagen exhibited thinly spread collagenous fibrous network. The thickness of the fibres were measured and found to be 10 m and 11 m for native and gallic acid treated collagen fibres, respectively. However, collagen treated with [Cr(GA)2 ]+ displayed the microfibril formation with thickness in nanometer level. The morphological change observed with respect to [Cr(GA)2 ] further supports the efficient crosslinking process by inhibiting the fibril formation compared to native and gallic acid treated collagen. 3.7. AFM imaging The changes in the secondary structure of collagen brought about by the metal complexes has been investigated by AFM imaging. AFM provides evidence for the self-assembly of collagen. The topological distribution and the interaction of metal ions onto the collagenous matrix can be explained using AFM images. AFM image of Type I collagen has many grooves and ridges in which collagen monomers are staggered into a quarterly fibrillar structure with a d-periodicity of 64 nm. The d-periodicity values observed for native
collagen, gallic acid and [Cr(GA)2 ] treated collagenous fibres are 62, 68.9 and 58.9 nm, respectively (Fig. 9). Gayathri et al., have shown a variation in d-periodicity of nearly 8 nm when collagen treated with various species of Cr(III) complexes compared to native collagen [30]. The observed marginal difference in the d-periodicity reveal that [Cr(GA)2 ]+ complex might displace the water involved in hydration network of the collagen, thereby resulting in structural rearrangement of collagen [30]. The slight variations in the d-period values indicate that no major structural alterations occurred. Similarly, the less difference in the d period values of gallic acid and [Cr(GA)2 ] treated collagen does not cause structural interruptions in the matrix. Slight loosening of triple helical configuration might have resulted in a decreased d period value of 58.9 nm. Also, the difference in d periodicity might be due to the displacement of water involved in the hydration network of collagen caused by the metal ions thereby affecting its intra and inter chemical bonds compared to gallic acid. 4. Conclusion Altogether, the data from the interaction of [Cr(GA)2 ] with collagen reveals that (i) the presence of Cr in the form of chromium complex did not alter the triple helical nature of the collagen; (ii) with [Cr(GA)2 ], the hydrothermal stability of the collagen improved by 6 ◦ C compared to gallic acid; (iii) the viscosity of collagen significantly reduced in the presence of complex; (iv) a significant delay in fibril formation by the complex; (v) SEM analysis confirms the inhibition of fibril formation in the presence of [Cr(GA)2 ] and (vi) reduction in d-periodicity of collagen with [Cr(GA)2 ]. The results from this study clearly provides the information on the presence
D. Jaikumar et al. / International Journal of Biological Macromolecules 96 (2017) 429–435
of Cr(III) in the form of polyphenolic complex i.e. [Cr(GA)2 ] further stabilizes collagen structure as well as crosslinking and might help in development of new products for leather tanning. Acknowledgments This research is supported by Science and Technology Revolution in Leather with a Green Touch (STRAIT) Institutional Project. V.G.V thanks DST-SERB for Ramanujan fellowship (SR/S2/RJN07/2011). The authors thank the reviewers for their valuable suggestions. References [1] S. Ricard-Blum, The collagen family, Cold Spring Harb. Perspect. Biol. 3 (1) (2011) a004978. [2] J. Kastelic, E. Baer, J.F.V Vincent, J.D. Currey (Eds.), Cambridge University Press, 397–435 (1980). [3] N.S. Demina, S.V. Lysenko, Microbiology 65 (1996) 257–265. [4] D.J. Harrington, Infect. Immunol. 64 (1996) 1885–1891. [5] J.R. Rao, R. Gayatri, R. Rajaram, B.U. Nair, T. Ramasami, Chromium(III) hydrolytic oligomers: their relevance to protein binding, Biochem. Biophys. Acta 1472 (3) (1999) 595–602. [6] H.Y. Shrivastava, B.U. Nair, J. Biomol. Struct. Dyn. 20 (2003) 575. [7] S. Ahmed, Arthritis Res. Ther. 12 (2010) 1–9. [8] A.A. Haroun, S.A. Toumy, J. Appl. Polym. Sci. 116 (2010) 770–776. [9] E. Haslam, Phytochemistry 68 (2007) 2713–2721. [10] B. Madhan, G. Krishnamoorthy, J.R. Rao, B.U. Nair, Role of green tea polyphenols in the inhibition of collagenolytic activity by collagenase, Int. J. Biol. Macromol. 41 (1) (2007) 16–22.
435
[11] J.C. Isenberg, D.T. Simionescu, N.R. Vyavahare, Biomaterials 26 (2005) 1237–1245. [12] M.E. Tedder, J. Liao, B. Weed, C. Stabler, H. Zhang, A. Simionescu, D.T. Simionescu, Tissue Eng. Part A 159 (2009) 1257–1268. [13] G. Krishnamoorthy, B. Madhan, S. Sadulla, J.R. Rao, M. Madhulatha, J. Appl. Polym. Sci. 108 (2008) 199–205. [14] G. Krishnamoorthy, R. Selvakumar, T.P. Sastry, S. Sadulla, A.B. Mandal, M. Doble, Mater. Sci. Eng. C 42 (2014) 164–171. [15] J.R. Rao, R. Gayatri, R. Rajaram, B.U. Nair, T. Ramasami, Biochem. Biophys. Acta 1472 (1999) 595–602. [16] E. Kong, S.J. Herzog, Inorg. Nucl. Chem. 32 (1970) 585–599. [17] G. Chandrakasan, D.A. Torchia, K.A. Piez, Preparation of intact monomeric collagen from rat tail tendon and skin and the structure of the nonhelical ends in solution, J. Biol. Chem. 251 (1976) 6062–6067. [18] N.N. Fathima, J.R. Rao, B.U. Nair, J. Inorg. Biochem. 100 (2006) 1774–1780. [19] H. Arakawa, M.-S. Tang, Chem. Res. Toxicol. 2 (2008) 1284–1289. [20] R. Borasky, G.C. Nutting, J. Am. Leather Chem. Assoc. 44 (1949) 830–841. [21] K.V. Srivatsan, R. Lakra, K. Purna Sai, M.S. Kiran, J. Mater. Chem. B 4 (2016) 1437–1447. [22] J.J. Lingane, R.L. Pecsok, Preparation of standard chromous sulfate or chromous chloride solutions of determinate concentration, Anal. Chem. 20 (5) (1948) 425–428. [23] M.L. Tiffany, S. Krim, Biopolymers 8 (1969) 347–359. [24] N. Greenfield, Nat. Protoc. 1 (2007) 2876–2890. [25] N.N. Fathima, A. Dhathathreyan, Int. J. Biol. Macromol. 45 (2009) 274–278. [26] Y. Li, Y. Li, Z. Du, G. Li, Thermochim. Acta 469 (2008) 71–76. [27] L. He, C. Mu, J. Shi, Q. Zhang, B. Shi, W. Lin, Modification of collagen with a natural cross-linker, procyanidin, Int. J. Biol. Macromol. 48 (2) (2011) 354–359. [28] R. Gayatri, R. Rajaram, B.U. Nair, F. Chandrasekaran, T. Ramasami, Proc. Indian Acad. Sci. 111 (1999) 133–145. [29] K.E. Kadler, D.F. Holmes, J.A. Trotter, J.A. Chapman, Biochem. J. 316 (1996) 1–11. [30] R. Gayatri, A.K. Sharma, R. Rajaram, T. Ramasami, Biochem. Biophys. Res. Commun. 283 (2001) 229–235.