- Email: [email protected]
Solution structure of cyanobacterial polysaccharide, sacran
Polymer 99 (2016) 767e770
Contents lists available at ScienceDirect
Polymer journal homepage: www.elsevier.com/locate/polymer
Solution structure of cyanobacterial polysaccharide, sacran Kazuhiro Shikinaka a, *, Kosuke Okeyoshi b, Hiroyasu Masunaga c, Maiko K. Okajima b, Tatsuo Kaneko b, ** a
Graduate School of Engineering, Tokyo University of Agriculture and Technology, Koganei 184-8588, Japan School of Materials Science, Japan Advanced Institute of Sciences and Technology (JAIST), Ishikawa 923-1292, Japan c Research & Utilization Division, SPring-8, Japan Synchrotron Radiation Research Institute, Hyogo 679e5198, Japan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 February 2016 Received in revised form 7 July 2016 Accepted 1 August 2016 Available online 3 August 2016
In this study, the solution structure of sacran, cyanobacterial polysaccharide, was estimated by synchrotron X-ray scattering. The chain conformation of sacran in solvent significantly differs depending on the concentration even though it has extraordinary stiffness (109 nm of persistent length). The sacran assemblies under concentrated conditions exhibited hierarchical structure (i.e., macroscopic liquid crystalline domain consisting of helices of sacran chains). The findings enable us to design sacran-based functional materials for various applications and to understand the molecular behavior of anionic biopolymers such as glycosaminoglycan and deoxyribonucleic acid. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Sacran Synchrotron X-ray scattering Solution structure
1. Introduction Sacran is a naturally-sulfated polysaccharide with an extremely high molecular weight Mw over 1 107 g/mol that is extracted from the jelly extracellular matrix of the freshwater cyanobacterium Aphanothece sacrum [1]. Various applications of sacran have been studied because of sacran's swelling property in water and its ability to absorb ions such as rare metals [2e5]. Recently, the potential of sacran for pharmaceutical applications based on its high absorption capability was also proposed [6]. Since sacran was used in the swollen state in the systems described above, understanding the chain conformation of sacran in solvent is important for designing materials with desired functions. The solution structure of sacran has been examined by rheological measurements, electrical conductivity tests, and dielectric relaxation behavior that are indirect evaluation methods for the solution structure such as chain stiffness and conformation [7]. When the solution is dried, a highlyoriented film showing anisotropic swelling in water is created [8]. The microscopic observation of sacran chains in a dried sample of a
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (T. Kaneko).
(K.
Shikinaka),
http://dx.doi.org/10.1016/j.polymer.2016.08.003 0032-3861/© 2016 Elsevier Ltd. All rights reserved.
[email protected]
solution was also performed [9]. Findings from these studies demonstrated the unique structural behavior of sacran chains in solvent. In addition, information about the chain conformation of sacran in the presence of water (i.e., no drying) is important not only in terms of understanding the solution behavior of rigid-rod anionic polysaccharides but also for considering the structure of the extracellular matrix of cyanobacterium which can be regarded as chromosome models. Furthermore, understanding the selforganizing behavior of sacran chains in solvent will assist in revealing the hierarchical organization mechanism of other polyanions in living systems such as glycosaminoglycan (GAG) and deoxyribonucleic acid (DNA). That is, sacran can be used as a structural model for these polyanions as all have extremely high molecular weights, many sulfate and carboxylic groups, and partial flexibility. In the present study, we performed synchrotron X-ray scattering of sacran solution by using a unique apparatus of tandem vertical undulator synchrotron radiation with high photon flux and an X-ray counting two dimensional pixel detector to estimate the solution structure of sacran directly. Here, we firstly revealed the persistent length of sacran chain and the detail chain conformation of sacran in the presence of solvent that are extraordinary one even relative to other biopolymers such as GAG and DNA.
768
K. Shikinaka et al. / Polymer 99 (2016) 767e770
cylinder model [13,14] given by following equation:
2. Experimental section 2.1. Materials
p
The aqueous LC solution of sacran, which was purchased from Green Science Materials Inc (Kumamoto, Japan), was prepared by mixing the sacran with ultra pure water (purified using a Milli-Q® Advantage A10® system) and heating at 80 C with agitation for 8 h to create a homogenous solution. The weight-average Mw and polydispersity of sacran were 2.0 107 g/mol and 1.2, respectively, as determined by gel permeation chromatography [1]. The absolute Mw and radius of gyration for sacran were 1.6 107 g/mol and 402 nm, respectively, as estimated from multi-angle laser light scattering [1]. 2.2. Synchrotron X-ray scattering The X-ray scattering measurements were performed using the SPring-8 (Hyogo, Japan) synchrotron orbital radiation beam line at BL45XU, which has a double-crystal diamond monochromator and K-B mirrors held at room temperature. The energy of the X-ray was 12.4 keV (wavelength, l ¼ 0.10 nm) and the beam size was 0.3 0.2 mm. All scattering experiments were performed on samples of sacran aqueous solutions at 25.0 C that were introduced into a quartz cell. The images of the scattering pattern were obtained at a frame size of 1475 1679 pixels and a pixel size of 172 172 mm using the X-ray photon counting two-dimensional pixel detector Pilatus3X 2 M [10]. The specimen-to-detector distance was 3.51 m. The data were corrected by background scattering from ultra pure water alone. The two-dimensional scattering patterns were circularly integrated and converted into onedimensional format (denoted as the scattering curve) using FIT2D software. 2.3. Estimation of scattering data From the X-ray scattering measurement, the q value was estimated according to Equation (3):
q ¼ 4p sinðq=2Þ=l
(3)
where q is the scattering angle. The scattering intensity I(q) is described by Equation (4) [11].
2 IðqÞ ¼ v20 rp rs f 2 SðqÞ
(4)
where rp and rs are the scattering length densities of sacran and the solvent, respectively; v0 is volume of sacran; and f is the singleparticle form factor [11]. Here, S(q), the structure factor, is given by Equation (5) in the range of 1/L < q < 1/D, where C is a constant; L and D are the characteristic length (e.g., Rg) and diameter of some structures formed by sacran, respectively; and df is the mass fractal dimension [12]. The second term in Equation (5) behaves as q-df, and therefore, the fractal dimension E is defined from the exponent in the relation I(q) ~ qE at 1/L < q < 1/D.
C df 1 G df 1 LE 1 þ q2 L2 1=2 SðqÞ ¼ 1 þ d=2 qL 1 þ q2 L2 h i sin df 1 arctanðqLÞ df 1
(5)
The relationship between I(q) and q were also evaluated by curve fitting according to theoretical scattering functions for the
scale2 PðqÞ ¼ Vcyl
p
Z2
f 2 ðq; aÞsin ada
0
j ðqr sin aÞ f ðq; aÞ ¼ 2 rcyl rsolv Vcyl j0 ðqH cos aÞ 1 ðqr sin aÞ Vcyl ¼ pr 2 L j0 ðxÞ ¼ sin x=x
(6)
Where J1 (x) is the first order Bessel function. a is defined as the angle between the cylinder axis and q. The integral over a averages the form factor over all possible orientations of the cylinder with respect to q. 3. Results and discussion In this study, synchrotron X-ray scattering of sacran solutions was performed to reveal the chain conformation of sacran in the swollen state. Here, we estimated the structure of sacran in pure water without some salts in which there is strong inter-chain electrostatic repulsion. In aqueous solution, sacran has many hydroxyl groups [6] that significantly weaken the signal-to-noise ratio of scattering. Furthermore, an extremely low overlap concentration C* of sacran (0.004 wt/v% [7]) due to its high Mw also prevents the detection of significant scattering from isolated sacran chain in solvent even under ordinary synchrotron radiation. To overcome this problem, we employed a tandem vertical undulator synchrotron beam apparatus with high photon flux as described in the Experimental section. Our experimental system produced significant scattering even when using a diluted sacran aqueous solution of 0.0025 wt/v% that is smaller than the C*. The X-ray scattering curves (i.e., the scattering intensity I(q) versus the scattering vector q) of sacran aqueous solution showed the unique structural behavior of sacran chains relative to ordinary polymers. Here, the conformation of sacran assemblies can be evaluated from the steepness of the slope of the curve defined by I(q) ~ qE (see Experimental section) [15,16]. Since the q ¼ 0.020e2.0 nm1 represents the real size of sacran assemblies, E ¼ 1.0, 2.0, and 3.0 in this region indicates the presence of rod-like, platelet-like, and network-like structures of sacran assemblies, respectively. As shown in Fig. 1, the E value was 3.0 at q ¼ 0.020e0.11 nm1 and 1.0 at q ¼ 0.11e2.0 nm1 in diluted sacran aqueous solution around C*. Since it has been revealed that the sacran chains near C* exist in an isolated state without intermolecular aggregation [7], E at q ¼ 0.020e0.11 nm1 indicates the presence of self-entangled (i.e., intramolecular aggregated) sacran chains as illustrated by the circle in Fig. 1. The E value changed from 3.0 to 1.0 at q ¼ 0.11 nm1 (57 nm), which corresponds to the thermic correlation length (TCL) [17]. E at q ¼ 0.11e2.0 nm1 showed an extraordinary straightness of the sacran chains relative to ordinary flexible polymers such as poly(ethylene glycol) in water (E ¼ 1.67) [18,19] due to strong interchain electrostatic repulsion. Since the network structure changes to an extended chain structure at q ¼ 0.11 nm1 without any intermediate state (e.g., blobs [17]), the persistent length (lp) of the sacran chains was described as lp ¼ TCL 6/p [20] that is higher value (109 nm at 0.0025 wt/v%) relative to a single-stranded anionic biopolymer than the lp values of alginate (~9 nm; polysaccharide) [21], fetal aggrecan (~21 nm; GAG) [22], and single-stranded DNA (~2.5 nm) [23]. The lp (i.e., TCL) value seems to be constant at various concentration as shown in Figs. 1e3. An enough electrostatic
K. Shikinaka et al. / Polymer 99 (2016) 767e770
Fig. 1. Scattering curve obtained from X-ray scattering of sacran aqueous solution at a concentration near C*. From the slope of the tangent lines, E was estimated. The closed dots are derived from Equation. (6) from ref. 13,14.
repulsion of the sacran chains even at their low concentration without some salt might cause the no concentration dependence of lp. The cylinder model function [13,14] well fit to X-ray scattering curve in which the chain diameter and the counter length are 1.76 nm and 1000 nm, respectively. These values are similar with our previous experimental results [1,9]. As shown in Fig. 2, the E value at q ¼ 0.020e0.11 nm1 was 2.0 in semi-diluted solution where an entanglement (i.e., intermolecular aggregation) of sacran chains occurs and is marked by the circle in Fig. 2 [7]. The E value at q ¼ 0.11e0.87 nm1 was 1.0, corresponding to the presence of rod-shaped structures at this scale. Contrastingly, at q ¼ 0.87e2.0 nm1, the E value changed to 2.0. Since a helical structure of sacran chains was recognized using electron microscopic images at this concentration range [9], it seems that these E value correspond to the helical structure of sacran assemblies as illustrated by the square in Fig. 2. With a further increase in the concentration of the aqueous sacran solution, the E value at q ¼ 0.020e0.11 nm1 changed to 3.0,
Fig. 2. Scattering curve obtained from X-ray scattering of sacran aqueous solution in the semi-diluted state. From the slope of the tangent lines, E was estimated.
769
Fig. 3. Scattering curve obtained from X-ray scattering of sacran aqueous solution in the concentrated state. From the slope of the tangent lines, E was estimated.
corresponding to the network formation of sacran chains that induces gelation of the solution (Fig. 3). From previous studies, it was also revealed that the sacran solution shows gelation above 0.25 wt/v% [9]. At this concentration range, clear peaks also emerged at q ¼ 0.23 nm1 (i.e., 27 nm) that indicated the formation of some regulated structure in the sacran assemblies. Since the gelated sacran solution exhibits macroscopic liquid crystalline (LC) domains with a length of micrometer ~ millimeter order and a diameter of submicron order that might consist of helices of sacran chains [9], it seems that the peaks correspond to the formation of LC domains by helices (i.e., bundling of helices). The origin of these peaks will be discussed the following sections. Here, the diameter r of sacran chains in their assemblies is estimated to be 1.76 nm from the cross-sectional plot [24] as shown in Fig. 4 according to the following equations:
qIðqÞ exp Rc ¼ r
ð1=2ÞR2c q2
. 20:5
(1)
(2)
where, Rc is root-square value from center-point. This r value is slightly larger than the combined diameter of two sacran chains (0.8e1.4 nm) [1]. Therefore, it seems that the sacran chains form a multi-helical structure as speculated in previous studies [9]. Since the r of helices in the sacran assemblies (1.76 nm) is much smaller than the size of the regulated structure (27 nm) suggested from the X-ray scattering results, the peak in the scattering curve should correspond to a pitch of helix in the LC domains of the sacran assemblies as shown in Fig. 5. The described loose helical structure of sacran chains in Fig. 5 is similar to the helix which was observed under transmission electron microscopic (TEM) images in Fig. 2 (b) of ref. [9]. The diameter and helical pitch found by TEM are larger than the values determined by X-ray scattering due to drying of the swollen sample under TEM observation. The nature of sacran as a stiff polysaccharide might induce such a loose association of sacran chains via a few moieties containing amino sugar residues in their chemical structure [1,9]. Thus, the presence of helices in the LC domain of sacran gel with highly regulated alignments was first verified from X-ray studies. The well-defined pitch of sacran helices might be induced by the well-regulated chemical structure (i.e., regulated interval of moieties such as amino sugar residues).
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K. Shikinaka et al. / Polymer 99 (2016) 767e770
other polysaccharides including GAGs [25e27] and DNA [28] (helical pitch of several nm) might be the result of the extraordinary stiffness and the few interaction moieties (amino groups) [9] of sacran chains. That is, the helical structure of anionic biopolymers might be determined by their persistence length and number of interaction groups. The present study is the first to show the chain conformations of sacran in the swollen state and the contribution of the chains in the process of hierarchical assembly. The obtained knowledge enables us to design sacran-based functional materials for industrial/ biomedical uses based on an understanding of their structural characteristics. Furthermore, the solution chemistry of sacran as a polyanion will allow for the creation of molecular nanoarchitectonics for the supramolecular assembly of anionic biopolymers such as GAGs and DNA that will improve our understanding of the scientific principles related to self-organization in living organisms. Acknowledgements
Fig. 4. Cross-section plot obtained from X-ray scattering of sacran aqueous solution at 0.25 wt/v%. From the slope of the red tangent, Rc was estimated according to Equation (1). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
4. Conclusion In conclusion, the structure of sacran chain in the swollen state was revealed by synchrotron X-ray scattering. The supramolecular structure of sacran chains completely differs depending on the concentration of aqueous solution. Surprisingly, sacran chains showed various shaped supramolecular architectures even though they have extraordinary stiffness (lp ¼ 109 nm). The lp value of sacran is larger than that of other single-stranded anionic biopolymers such as GAGs (~21 nm) [22] and single-stranded DNA (~2.5 nm) [23] despite their homogenous chemical structure. The lp values of GAGs and DNA might be estimated low relative to their true values due to their low molecular weight (<106 g/mol) that is further reduced until extracted processes from the living organisms. Furthermore, the helical structure (i.e., diameter and pitch) in the LC domains of gelated sacran solution that had been the subject of speculation in various studies [7,9] was also revealed in detail. The loose helices of sacran chains (helical pitch of 27 nm) relative to
Fig. 5. Schematic illustration of the helical structure in the LC domain of sacran assemblies in the concentrated state.
The synchrotron orbital radiation experiments were performed at BL45XU in SPring-8 with the approval of the Japan Synchrotron Radiation Research Institute (JASRI) (Proposal No. 2015B1251). This work was supported by Grant-in-Aid for Young Scientists B (16K17956 and 26870179) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. References [1] M.K. Okajima, T. Bamba, Y. Kaneso, K. Hirata, E. Fukusaki, S. Kajiyama, T. Kaneko, Macromolecules 41 (2008) 4061e4064. [2] M.K. Okajima, S. Miyazato, T. Kaneko, Langmuir 25 (2009) 8526e8531. [3] M.K. Okajima, M. Nakamura, T. Mitsumata, T. Kaneko, Biomacromolecules 11 (2010) 1773e1776. [4] M.K. Okajima, T. Higashi, R. Asakawa, T. Mitsumata, D. Kaneko, T. Kaneko, T. Ogawa, H. Kurata, S. Isoda, Biomacromolecules 11 (2010) 3172e3177. [5] M.K. Okajima, Q.T.I. Nguyen, M. Nakamura, T. Ogawa, H. Kurata, T. Kaneko, J. Appl, Polym. Sci. 128 (2013) 676e683. [6] K. Motoyama, Y. Tanida, K. Hata, T. Hayashi, T. Higashi, Y. Ishitsuka, Y. Kondo, T. Irie S. Kaneko, H. Arima, Chem. Pharm. Bull. 62 (2014) 636e641. [7] T. Mitsumata, T. Miutra, N. Takahashi, M. Kawai, M.K. Okajima, T. Kaneko, Phys. Rev. E 87 (2013) 042607. [8] M. Okajima, R. Mishima, K. Amornwachirabodee, K. Okeyoshi, T. Kaneko, RSC Adv. 5 (2015) 86723e86729. [9] M.K. Okajima, D. Kaneko, T. Mitsumata, T. Kaneko, J. Watanabe, Macromolecules 42 (2009) 3057e3062. €nnimanna, R. Baurb, E.F. Eikenberrya, S. Kohouta, M. Lindnerc, [10] C. Bro B. Schmitta, R. Horisberger, Nucl. Inst. Meth. Phys. Res. A 465 (2001) 235e239. [11] T. Freltoft, J.K. Kjems, S.K. Sinha, Phys. Rev. B 33 (1986) 269e275. [12] J. Teixeira, J. Appl. Crystallogr. 21 (1998) 781e785. [13] N. Saito, Y. Ikeda, J. Phys. Soc. Jpn. 7 (1952) 227. [14] A. Guinier, G. Fournet, Small-angle Scattering of X-rays, John Wiley and Sons, New York, 1955. [15] G. Porod, Small-angle X-ray Scattering, Academic Press, London, UK, 1982, pp. 17e51. [16] P.W. Schmidt, The Fractal Approach to Heterogeneous Chemistry, John Wiley & Sons, New York, 1989. [17] G.R. Strobl, The Physics of Polymer, third ed, Springer, Berlin, 2007. [18] M. Rubinstein, R.H. Colby, Polymer Physics, Oxford University Press, Oxford, 2003. [19] K. Mayumi, K. Ito, Polymer 51 (2010) 959e967. [20] A. Michelman-Ribeiro, H. Boukari, R. Nossal, F. Horkay, Macromolecules 37 (2004) 10212e10214. [21] B.T. Stokke, D.A. Brant, Biopolymers 30 (1990) 1161e1181. [22] L. Ng, A.J. Grodzinsky, P. Patwari, J. Sandy, A. Plaas, C. Ortiz, J. Struct. Biol. 143 (2003) 242e257. [23] H. Chen, S.P. Meisburger, S.A. Pabit, J.L. Sutton, W.W. Webb, L. Pollack, Proc. Nat. Acad. Sci. U. S. A. 109 (2012) 799e804. [24] P. Mittelbach, Acta Phys. Austriaca 19 (1964) 53e102. [25] N.S. Anderson, J.W. Campbell, M.M. Harding, D.A. Rees, J.W.B. Samuel, J. Mol. Biol. 45 (1969) 85e97. [26] S. Dumitriu, Polysaccharides: Structural Diversity and Functional Bersatility, second ed., CRC Press, US, 2004. [27] S. Arnott, D.A. Rees, E.R. Morris, Molecular Biophysics of the Extracellular Matrix, Springer, 2012. Berlin. [28] J.D. Watson, F.H.C. Crick, Nature 171 (1953) 737e738.
Contents lists available at ScienceDirect
Polymer journal homepage: www.elsevier.com/locate/polymer
Solution structure of cyanobacterial polysaccharide, sacran Kazuhiro Shikinaka a, *, Kosuke Okeyoshi b, Hiroyasu Masunaga c, Maiko K. Okajima b, Tatsuo Kaneko b, ** a
Graduate School of Engineering, Tokyo University of Agriculture and Technology, Koganei 184-8588, Japan School of Materials Science, Japan Advanced Institute of Sciences and Technology (JAIST), Ishikawa 923-1292, Japan c Research & Utilization Division, SPring-8, Japan Synchrotron Radiation Research Institute, Hyogo 679e5198, Japan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 February 2016 Received in revised form 7 July 2016 Accepted 1 August 2016 Available online 3 August 2016
In this study, the solution structure of sacran, cyanobacterial polysaccharide, was estimated by synchrotron X-ray scattering. The chain conformation of sacran in solvent significantly differs depending on the concentration even though it has extraordinary stiffness (109 nm of persistent length). The sacran assemblies under concentrated conditions exhibited hierarchical structure (i.e., macroscopic liquid crystalline domain consisting of helices of sacran chains). The findings enable us to design sacran-based functional materials for various applications and to understand the molecular behavior of anionic biopolymers such as glycosaminoglycan and deoxyribonucleic acid. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Sacran Synchrotron X-ray scattering Solution structure
1. Introduction Sacran is a naturally-sulfated polysaccharide with an extremely high molecular weight Mw over 1 107 g/mol that is extracted from the jelly extracellular matrix of the freshwater cyanobacterium Aphanothece sacrum [1]. Various applications of sacran have been studied because of sacran's swelling property in water and its ability to absorb ions such as rare metals [2e5]. Recently, the potential of sacran for pharmaceutical applications based on its high absorption capability was also proposed [6]. Since sacran was used in the swollen state in the systems described above, understanding the chain conformation of sacran in solvent is important for designing materials with desired functions. The solution structure of sacran has been examined by rheological measurements, electrical conductivity tests, and dielectric relaxation behavior that are indirect evaluation methods for the solution structure such as chain stiffness and conformation [7]. When the solution is dried, a highlyoriented film showing anisotropic swelling in water is created [8]. The microscopic observation of sacran chains in a dried sample of a
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (T. Kaneko).
(K.
Shikinaka),
http://dx.doi.org/10.1016/j.polymer.2016.08.003 0032-3861/© 2016 Elsevier Ltd. All rights reserved.
[email protected]
solution was also performed [9]. Findings from these studies demonstrated the unique structural behavior of sacran chains in solvent. In addition, information about the chain conformation of sacran in the presence of water (i.e., no drying) is important not only in terms of understanding the solution behavior of rigid-rod anionic polysaccharides but also for considering the structure of the extracellular matrix of cyanobacterium which can be regarded as chromosome models. Furthermore, understanding the selforganizing behavior of sacran chains in solvent will assist in revealing the hierarchical organization mechanism of other polyanions in living systems such as glycosaminoglycan (GAG) and deoxyribonucleic acid (DNA). That is, sacran can be used as a structural model for these polyanions as all have extremely high molecular weights, many sulfate and carboxylic groups, and partial flexibility. In the present study, we performed synchrotron X-ray scattering of sacran solution by using a unique apparatus of tandem vertical undulator synchrotron radiation with high photon flux and an X-ray counting two dimensional pixel detector to estimate the solution structure of sacran directly. Here, we firstly revealed the persistent length of sacran chain and the detail chain conformation of sacran in the presence of solvent that are extraordinary one even relative to other biopolymers such as GAG and DNA.
768
K. Shikinaka et al. / Polymer 99 (2016) 767e770
cylinder model [13,14] given by following equation:
2. Experimental section 2.1. Materials
p
The aqueous LC solution of sacran, which was purchased from Green Science Materials Inc (Kumamoto, Japan), was prepared by mixing the sacran with ultra pure water (purified using a Milli-Q® Advantage A10® system) and heating at 80 C with agitation for 8 h to create a homogenous solution. The weight-average Mw and polydispersity of sacran were 2.0 107 g/mol and 1.2, respectively, as determined by gel permeation chromatography [1]. The absolute Mw and radius of gyration for sacran were 1.6 107 g/mol and 402 nm, respectively, as estimated from multi-angle laser light scattering [1]. 2.2. Synchrotron X-ray scattering The X-ray scattering measurements were performed using the SPring-8 (Hyogo, Japan) synchrotron orbital radiation beam line at BL45XU, which has a double-crystal diamond monochromator and K-B mirrors held at room temperature. The energy of the X-ray was 12.4 keV (wavelength, l ¼ 0.10 nm) and the beam size was 0.3 0.2 mm. All scattering experiments were performed on samples of sacran aqueous solutions at 25.0 C that were introduced into a quartz cell. The images of the scattering pattern were obtained at a frame size of 1475 1679 pixels and a pixel size of 172 172 mm using the X-ray photon counting two-dimensional pixel detector Pilatus3X 2 M [10]. The specimen-to-detector distance was 3.51 m. The data were corrected by background scattering from ultra pure water alone. The two-dimensional scattering patterns were circularly integrated and converted into onedimensional format (denoted as the scattering curve) using FIT2D software. 2.3. Estimation of scattering data From the X-ray scattering measurement, the q value was estimated according to Equation (3):
q ¼ 4p sinðq=2Þ=l
(3)
where q is the scattering angle. The scattering intensity I(q) is described by Equation (4) [11].
2 IðqÞ ¼ v20 rp rs f 2 SðqÞ
(4)
where rp and rs are the scattering length densities of sacran and the solvent, respectively; v0 is volume of sacran; and f is the singleparticle form factor [11]. Here, S(q), the structure factor, is given by Equation (5) in the range of 1/L < q < 1/D, where C is a constant; L and D are the characteristic length (e.g., Rg) and diameter of some structures formed by sacran, respectively; and df is the mass fractal dimension [12]. The second term in Equation (5) behaves as q-df, and therefore, the fractal dimension E is defined from the exponent in the relation I(q) ~ qE at 1/L < q < 1/D.
C df 1 G df 1 LE 1 þ q2 L2 1=2 SðqÞ ¼ 1 þ d=2 qL 1 þ q2 L2 h i sin df 1 arctanðqLÞ df 1
(5)
The relationship between I(q) and q were also evaluated by curve fitting according to theoretical scattering functions for the
scale2 PðqÞ ¼ Vcyl
p
Z2
f 2 ðq; aÞsin ada
0
j ðqr sin aÞ f ðq; aÞ ¼ 2 rcyl rsolv Vcyl j0 ðqH cos aÞ 1 ðqr sin aÞ Vcyl ¼ pr 2 L j0 ðxÞ ¼ sin x=x
(6)
Where J1 (x) is the first order Bessel function. a is defined as the angle between the cylinder axis and q. The integral over a averages the form factor over all possible orientations of the cylinder with respect to q. 3. Results and discussion In this study, synchrotron X-ray scattering of sacran solutions was performed to reveal the chain conformation of sacran in the swollen state. Here, we estimated the structure of sacran in pure water without some salts in which there is strong inter-chain electrostatic repulsion. In aqueous solution, sacran has many hydroxyl groups [6] that significantly weaken the signal-to-noise ratio of scattering. Furthermore, an extremely low overlap concentration C* of sacran (0.004 wt/v% [7]) due to its high Mw also prevents the detection of significant scattering from isolated sacran chain in solvent even under ordinary synchrotron radiation. To overcome this problem, we employed a tandem vertical undulator synchrotron beam apparatus with high photon flux as described in the Experimental section. Our experimental system produced significant scattering even when using a diluted sacran aqueous solution of 0.0025 wt/v% that is smaller than the C*. The X-ray scattering curves (i.e., the scattering intensity I(q) versus the scattering vector q) of sacran aqueous solution showed the unique structural behavior of sacran chains relative to ordinary polymers. Here, the conformation of sacran assemblies can be evaluated from the steepness of the slope of the curve defined by I(q) ~ qE (see Experimental section) [15,16]. Since the q ¼ 0.020e2.0 nm1 represents the real size of sacran assemblies, E ¼ 1.0, 2.0, and 3.0 in this region indicates the presence of rod-like, platelet-like, and network-like structures of sacran assemblies, respectively. As shown in Fig. 1, the E value was 3.0 at q ¼ 0.020e0.11 nm1 and 1.0 at q ¼ 0.11e2.0 nm1 in diluted sacran aqueous solution around C*. Since it has been revealed that the sacran chains near C* exist in an isolated state without intermolecular aggregation [7], E at q ¼ 0.020e0.11 nm1 indicates the presence of self-entangled (i.e., intramolecular aggregated) sacran chains as illustrated by the circle in Fig. 1. The E value changed from 3.0 to 1.0 at q ¼ 0.11 nm1 (57 nm), which corresponds to the thermic correlation length (TCL) [17]. E at q ¼ 0.11e2.0 nm1 showed an extraordinary straightness of the sacran chains relative to ordinary flexible polymers such as poly(ethylene glycol) in water (E ¼ 1.67) [18,19] due to strong interchain electrostatic repulsion. Since the network structure changes to an extended chain structure at q ¼ 0.11 nm1 without any intermediate state (e.g., blobs [17]), the persistent length (lp) of the sacran chains was described as lp ¼ TCL 6/p [20] that is higher value (109 nm at 0.0025 wt/v%) relative to a single-stranded anionic biopolymer than the lp values of alginate (~9 nm; polysaccharide) [21], fetal aggrecan (~21 nm; GAG) [22], and single-stranded DNA (~2.5 nm) [23]. The lp (i.e., TCL) value seems to be constant at various concentration as shown in Figs. 1e3. An enough electrostatic
K. Shikinaka et al. / Polymer 99 (2016) 767e770
Fig. 1. Scattering curve obtained from X-ray scattering of sacran aqueous solution at a concentration near C*. From the slope of the tangent lines, E was estimated. The closed dots are derived from Equation. (6) from ref. 13,14.
repulsion of the sacran chains even at their low concentration without some salt might cause the no concentration dependence of lp. The cylinder model function [13,14] well fit to X-ray scattering curve in which the chain diameter and the counter length are 1.76 nm and 1000 nm, respectively. These values are similar with our previous experimental results [1,9]. As shown in Fig. 2, the E value at q ¼ 0.020e0.11 nm1 was 2.0 in semi-diluted solution where an entanglement (i.e., intermolecular aggregation) of sacran chains occurs and is marked by the circle in Fig. 2 [7]. The E value at q ¼ 0.11e0.87 nm1 was 1.0, corresponding to the presence of rod-shaped structures at this scale. Contrastingly, at q ¼ 0.87e2.0 nm1, the E value changed to 2.0. Since a helical structure of sacran chains was recognized using electron microscopic images at this concentration range [9], it seems that these E value correspond to the helical structure of sacran assemblies as illustrated by the square in Fig. 2. With a further increase in the concentration of the aqueous sacran solution, the E value at q ¼ 0.020e0.11 nm1 changed to 3.0,
Fig. 2. Scattering curve obtained from X-ray scattering of sacran aqueous solution in the semi-diluted state. From the slope of the tangent lines, E was estimated.
769
Fig. 3. Scattering curve obtained from X-ray scattering of sacran aqueous solution in the concentrated state. From the slope of the tangent lines, E was estimated.
corresponding to the network formation of sacran chains that induces gelation of the solution (Fig. 3). From previous studies, it was also revealed that the sacran solution shows gelation above 0.25 wt/v% [9]. At this concentration range, clear peaks also emerged at q ¼ 0.23 nm1 (i.e., 27 nm) that indicated the formation of some regulated structure in the sacran assemblies. Since the gelated sacran solution exhibits macroscopic liquid crystalline (LC) domains with a length of micrometer ~ millimeter order and a diameter of submicron order that might consist of helices of sacran chains [9], it seems that the peaks correspond to the formation of LC domains by helices (i.e., bundling of helices). The origin of these peaks will be discussed the following sections. Here, the diameter r of sacran chains in their assemblies is estimated to be 1.76 nm from the cross-sectional plot [24] as shown in Fig. 4 according to the following equations:
qIðqÞ exp Rc ¼ r
ð1=2ÞR2c q2
. 20:5
(1)
(2)
where, Rc is root-square value from center-point. This r value is slightly larger than the combined diameter of two sacran chains (0.8e1.4 nm) [1]. Therefore, it seems that the sacran chains form a multi-helical structure as speculated in previous studies [9]. Since the r of helices in the sacran assemblies (1.76 nm) is much smaller than the size of the regulated structure (27 nm) suggested from the X-ray scattering results, the peak in the scattering curve should correspond to a pitch of helix in the LC domains of the sacran assemblies as shown in Fig. 5. The described loose helical structure of sacran chains in Fig. 5 is similar to the helix which was observed under transmission electron microscopic (TEM) images in Fig. 2 (b) of ref. [9]. The diameter and helical pitch found by TEM are larger than the values determined by X-ray scattering due to drying of the swollen sample under TEM observation. The nature of sacran as a stiff polysaccharide might induce such a loose association of sacran chains via a few moieties containing amino sugar residues in their chemical structure [1,9]. Thus, the presence of helices in the LC domain of sacran gel with highly regulated alignments was first verified from X-ray studies. The well-defined pitch of sacran helices might be induced by the well-regulated chemical structure (i.e., regulated interval of moieties such as amino sugar residues).
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other polysaccharides including GAGs [25e27] and DNA [28] (helical pitch of several nm) might be the result of the extraordinary stiffness and the few interaction moieties (amino groups) [9] of sacran chains. That is, the helical structure of anionic biopolymers might be determined by their persistence length and number of interaction groups. The present study is the first to show the chain conformations of sacran in the swollen state and the contribution of the chains in the process of hierarchical assembly. The obtained knowledge enables us to design sacran-based functional materials for industrial/ biomedical uses based on an understanding of their structural characteristics. Furthermore, the solution chemistry of sacran as a polyanion will allow for the creation of molecular nanoarchitectonics for the supramolecular assembly of anionic biopolymers such as GAGs and DNA that will improve our understanding of the scientific principles related to self-organization in living organisms. Acknowledgements
Fig. 4. Cross-section plot obtained from X-ray scattering of sacran aqueous solution at 0.25 wt/v%. From the slope of the red tangent, Rc was estimated according to Equation (1). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
4. Conclusion In conclusion, the structure of sacran chain in the swollen state was revealed by synchrotron X-ray scattering. The supramolecular structure of sacran chains completely differs depending on the concentration of aqueous solution. Surprisingly, sacran chains showed various shaped supramolecular architectures even though they have extraordinary stiffness (lp ¼ 109 nm). The lp value of sacran is larger than that of other single-stranded anionic biopolymers such as GAGs (~21 nm) [22] and single-stranded DNA (~2.5 nm) [23] despite their homogenous chemical structure. The lp values of GAGs and DNA might be estimated low relative to their true values due to their low molecular weight (<106 g/mol) that is further reduced until extracted processes from the living organisms. Furthermore, the helical structure (i.e., diameter and pitch) in the LC domains of gelated sacran solution that had been the subject of speculation in various studies [7,9] was also revealed in detail. The loose helices of sacran chains (helical pitch of 27 nm) relative to
Fig. 5. Schematic illustration of the helical structure in the LC domain of sacran assemblies in the concentrated state.
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