Biomacromolecules 2004, 5, 186-196
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Galactose-Substituted Alginate 2: Conformational Aspects Ivan Donati,* Anna Coslovi, Amelia Gamini, Gudmund Skjåk-Bræk,‡ Amedeo Vetere, Cristiana Campa,† and Sergio Paoletti Department of Biochemistry, Biophysics and Macromolecular Chemistry, University of Trieste, Via Licio Giorgieri 1, I-34127 Trieste, Italy Received August 21, 2003
Galactose moieties have been introduced on the uronic groups of alginates from different sources via an N-glycosidic bond, thus affecting the net charge on the polymer chain. The modified polymers have been analyzed by means of viscosity and of high-performance size-exclusion chromatography combined with refractive index multiple angle laser light scattering (HPSEC-RI-MALLS) measurements. The latter technique enabled us to determine the molecular weight of the modified polymers, proving that the synthetic procedure did not affect the chemical integrity of the chain. The intrinsic viscosity and the radius of gyration data showed that the hydrodynamic properties of the polymer chain varied with the degree and the pattern of substitution. In the presence of a relatively low galactose content (up to 19%), a decrease of the hydrodynamic dimensions of the coil was experienced, while on increasing the degree of substitution (especially on GG diads) a re-extension of the chain was discovered. Measurements of intrinsic viscosity at different values of the degree of dissociation have demonstrated that this effect cannot be solely explained by the reduction of the charge density of the polymer. Rather, it implies the occurrence of conformational changes of the chain that are specific to the chemical nature of the site of substitution. These data have been supported by the values of the persistence length of the natural and modified polymers obtained with the Doty-Benoit equation. The chiro-optical properties of the modified polymers studied by means of circular dichroism (CD) spectroscopy confirmed that conformational variations occurred to the polymeric chain upon introduction of galactose residues. Introduction Polysaccharides constitute major components of that part of the biological scenery, which is often cumulatively called the “extracellular matrix” (ECM). Together with the whole matrix biopolymers, they augment the mechanical stability through the formation of a three-dimensional network, ensure appropriate dynamic response to stresses, and create highly swollen environments with controlled permeability. Moreover, matrix biopolymers participate in the immunological “intelligence” network involved in cell/cell and guest/host specific interactions, control the tissue structure, regulate the function of cells, and allow the diffusion of nutrients, metabolites, and growth factors.1,2 Therefore, polysaccharides from different sources (e.g., hyaluronate, alginate, and chitosan to mention only a few) represent appealing candidates to obtain three-dimensional scaffolds, typically hydrogels, acting as analogues of the natural extracellular matrix. Alginates are a family of polysaccharides produced by brown algae3 and bacteria.4,5 Chemically, they are linear copolymers of 1 f 4-linked β-D-mannuronic acid (M) and * Corresponding author. Present address: Institute of BiotechnologyNTNU, Sem Sælands vei 6-8, N-7491 Trondheim, Norway. E-mail:
[email protected]. ‡ On leave of absence from Institute of Biotechnology, Norwegian University of Science and Technology (NTNU), Sem Sælands vei 6-8, N-7491 Trondheim, Norway. † Present permanent address: Bracco Imaging, CRM-TS, AREA Science Park, Building Q, SS14 Km 163.5, I-34012 Basovizza (TS).
R-L-guluronic acid (G). The composition and sequential arrangement of the two residues varies with the species or the tissue from which they are isolated.6 The monomers are arranged in a blockwise pattern along the chain with homopolymeric regions of M (M-blocks) and G (G-blocks) residues interspersed with regions of alternating structure (MG-blocks). Divalent cations such as calcium, strontium, and barium bind preferentially to the G-blocks in a highly cooperative manner, accounting for the formation of ionic hydrogels via the so-called “egg-box” model.7 The rapid gel formation in the presence of millimolar concentrations of calcium ions, as well as the elucidation of the structure-function relationships, has established alginate as a very versatile material for preparing microcapsules for cell therapy. Different cells have been suggested as candidates for gel immobilization including parathyroid cells for treatment of hypocalcemia,8 dopamine-producing adrenal chromaffin cells for treatment of Parkinson’s disease,9 and endostatin-producing cells for treatment of brain tumors.10 Major interest has been focused on insulinproducing cells for the treatment of type 1 diabetes, and alginate-poly-L-lysine capsules containing pancreatic islets of Langerhans have been shown to reverse diabetes in large animals.11 It is important to underline that in all of the reported examples the main goal of the gel is to act as a barrier between the transplanted cells and the immune system of the host. No specific interaction between the polysaccharide
10.1021/bm030063k CCC: $27.50 © 2004 American Chemical Society Published on Web 12/03/2003
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Galactose-Substituted Alginate 2
Table 1. Extent and Pattern of Galactose Substitution for Alginate from L. hyperborea and M. pyrifera Sources as Determined by 1H NMR sample
polymer
ds (%)a
Gal-G (%)b
Gal-M (%)c
Gal-G-(M) (%)d
Gal-G-(G) (%)e
Gal-M-(G) (%)f
Gal-M-(M) (%)g
1 2 3 4 5 6 7
L. hyperborea L. hyperborea L. hyperborea L. hyperborea M. pyrifera M. pyrifera M. pyrifera
0 7 19 36 0 16 38
0 7 19 29 0 12 20
0 h h 7 0 4 18
0 7 7.2 7.3 0 8 8
0 h 11.8 21.7 0 4 12
0 h h 7 0 4 9
0 h h h 0 h ∼9
a Total amount of galactose introduced on alginate chain. b Amount of galactose introduced on G residues: (Gal-G) + (Gal-M) ) ds. c Amount of galactose introduced on M residues: (Gal-G) + (Gal-M) ) ds. d Derivatization on a G residue neighboring an M residue: (Gal-G-(M)) + (Gal-G-(G)) ) Gal-G. e Derivatization on a G residue neighboring a G residue: (Gal-G-(M)) + (Gal-G-(G)) ) Gal-G. f Derivatization on an M residue neighboring a G residue: (Gal-M-(G)) + (Gal-M-(M)) ) Gal-M. g Derivatization on an M residue neighboring an M residue: (Gal-M-(G)) + (Gal-M-(M)) ) Gal-M. h Not determined (below the detection limit).
and the cells takes place because alginate is known not to be a bioadhesive material.12 In recent years, many efforts have been directed in the field of the so-called “polymer engineering”. This term usually refers to the modification of the polymer chain introducing cell-specific ligands or extracellular signaling molecules, such as peptides and oligosaccharides. These “smart” materials, which should therefore be able to directly intervene in the cell-cell and cell-ECM recognition processes controlling adhesion, growth, and differentiation of the cells,13 as well as structure and functions of the engineered tissue formed,14,15 are usually addressed as thirdgeneration biomaterials.16,17 On the basis of these considerations and prompted by the appealing gel-forming properties of alginate, Mooney and co-workers introduced an RGDcontaining cell adhesion ligand on alginate. Such a “smart” material has been demonstrated to provide for the adhesion, proliferation, and differentiation of skeletal myoblasts.18,19 On a similar basis, Akaike and co-workers succeeded in maintaining liver functions in hepatocytes encapsulated in a galactose-substituted alginate.20 Despite the great attention directed toward the evaluation of the biological effect of the engineered materials on cells, the consequences of the chemical modification on the physical-chemical properties of the polysaccharide have not been completely disclosed. Although this can be regarded as a trivial question, the knowledge of the conformational behavior in solution and of the physical properties of polysaccharides is needed to choose the best material for biomedical applications. Just to mention one, the variation of composition of different alginate samples, resulting in differences in related hydrodynamic properties,21 causes the enormous differences between alginate from Laminaria hyperborea, a perfectly biocompatible material,22 and that from a Pseudomonas source, an active pathogenic agent in cystic fibrosis.23 In addition, it has been observed that the presence of acetyl groups is related to fundamental steps in the bacterial alginate biosynthesis and its in vivo modification, providing the organism with a mechanism for controlling the degree of epimerization of the exocellular polysaccharide and therefore its physical properties such as the gelforming ability.24 Moreover, even a small percentage of acetyl groups was demonstrated to be able to modify the conformational behavior of a bacterial alginate.25 We have previously reported on the synthesis and the gelling properties of a novel galactose-substituted alginate.26
In the present paper, we deal with the conformational characterization of the modified polymers. In particular, the conformational effects of the introduction of galactose moieties on the uronic residues have been analyzed considering the intrinsic viscosity, the radius of gyration, and chirooptical properties by CD. Furthermore, an evaluation of the stiffness of the galactose-substituted alginates has been obtained by calculating the value of their persistence length. Materials and Methods A commercial sample of sodium alginate isolated from Laminaria hyperborea stipe, LF 10/60, (FG ) 0.69; FGG ) 0.56)27 and from Macrocystis pyrifera (FG ) 0.42; FGG ) 0.20)27 were provided by Protan A/S (Norway). 1-Ethyl-3[3-(dimethylamino)-propyl]carbodiimide hydrochloride (EDC) and sodium chloride were purchased from Aldrich Chemical Co. (Milwaukee, WI). N-Hydroxysuccinimide (NHS) and 2-[N-morpholino]ethanesulfonic acid were purchased from Sigma Chemical Co. (St. Louis, MO). Synthesis of Galactose-Substituted Alginate. The synthesis was carried out as previously reported26 coupling alginate with 1-amino-1-deoxy-β-D-galactose28,29 in the presence of NHS and EDC in MES buffer. The modified polymers have been extensively dialyzed against deionized water, the pH was increased to about 7.2 by addition of dilute NaOH, and the polymers were freeze-dried. The galactosesubstituted polymers obtained were analyzed by means of 1 H NMR to establish both the extent and the pattern of substitution.26 Table 1 reports the results of the 1H NMR analysis for products 1-7. It is noteworthy that, as previously reported,26 as the frequency of alternating sequences increases, for example, in the alginate from M. pyrifera, the reactivity toward the 1-amino-1-deoxy-galactose increases and therefore the total degree of substitution increases. 1 H NMR Spectroscopy. Samples were prepared as described by Grasdalen et al.30 The 1H NMR spectra were recorded at 90 °C with Bruker WM 300. The chemical shifts are expressed in ppm downfield from the signal for 3-(trimethylsilyl)propanesulfonate. Viscosity Measurements. Reduced capillary viscosity of the sodium form of samples listed in Table 1 was measured in 0.1 M NaCl at 25 °C by using a Schott-Gera¨te AVS/G automatic apparatus and an Ubbelohde type viscometer. Intrinsic viscosity values were determined therefrom by analyzing the concentration dependence of the reduced
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specific viscosity (ηsp/c) and the reduced logarithm of the relative viscosity (ln ηrel/c) by using the Huggins (eq 1) and Kraemer (eq 2) equations, respectively. ηsp/c ) [η] + k′[η]2c
(1)
(ln ηrel)/c ) [η] - k′′[η]2c
(2)
where k′ and k′′ are the Huggins and Kraemer constants, respectively. For samples 5, 6, and 7, the Smidsrød B-value31 was determined measuring intrinsic viscosity at different sodium chloride concentrations at 25 °C. Experiments at Different Values of Polymer Charge Density. Viscosity measurements were performed in 0.1 M NaCl at 25 °C on unsubstituted alginates at different values of polymer charge density by controlling the value of the degree of ionization R, defined as R ) RN +
10-pH 10-pOH Cp Cp
(3)
where RN, the degree of neutralization, is the equivalent ratio between the (formally) added base and the total carboxylic groups. For conditions around neutrality, obviously R ) RN, whereas for acidic pH values, it holds that R ) RN +
10-pH Cp
(4)
The polymer concentration, Cp, is given in monomol/L, that is, in moles of monomer uronic acid, irrespective of the chemical identity, per liter. High-Performance Size-Exclusion Chromatography Combined with Multiple Angle Laser Light Scattering (HPSEC-RI-MALLS). The HPSEC-RI-MALLS system consisted of an online degasser (Shimadzu DGU-4A), a pump (ShimadzuLC-10AD), and three serially connected columns (TSK gel G6000/5000/4000 PWXL). The eluent was 0.05 M Na2SO4 with 0.01 M EDTA (pH 6) at 0.5 mL/min. Detectors were refractive index (RI), UV monitor (Pharmacia LKB UV-M II, Amersham Pharmacia Biotech., Uppsala, Sweden), and multiple angle laser light scattering (MALLS, Dawn DSP equipped with a He-Ne laser, 632.8 nm, Wyatt Technology Corp., Santa Barbara, CA). Samples were dissolved at a concentration of ≈1 mg/mL in 0.05 M Na2SO4 with 0.01 M EDTA, pH 6, and filtered through 0.22 µm filters before injection of 100 µL. Data for molecular weight determination and conformation were analyzed using ASTRA software (version 4.70.07, Wyatt Technology Corp., Santa Barbara, CA). The refractive index increment (dn/dc) used for alginate was 0.15, as reported by Mackie and coworkers.32 The angular fit was based on the Debye procedure; weight-average molecular weight, Mw, and number-average molecular weight, Mn, were obtained following a first-order polynomial curve fitting of log M (M ) molecular weight) versus elution volume. Mw and the z-average radius of gyration 〈sz2〉1/2 were obtained from unfitted data, and no additional data smoothing was applied according to the manual.
Table 2. Weight-Average Molecular Weight and Radius of Gyration for Samples 1-7 sample
Mw(exptl)a (×103 g/mol)
1 2 3 4 5 6 7
126 133 137 147 213 260 291
Mw(calcd)b (×103 g/mol) 132 143 158 237 270
IP (Mw/Mn)
IP′ (Mz/Mn)
〈sz2〉1/2 (nm)c
1.76 1.90 1.83 1.94 2.01 2.31 1.96
3.06 3.19 4.46 4.38 3.94 5.52 3.85
56.9 50.6 43.7 44.2 67.1 56.8 60.9
a Weight-average molecular weight as determined by HPSEC-RIMALLS. b Weight-average molecular weight calculated from the extent of galactose introduced on the polymer chain on the basis of the weightaverage molecular weight of native alginate samples. c Radius of gyration as determined by HPSEC-RI-MALLS.
Circular Dichroism (CD). Circular dichroism spectra of the sodium form of the polymers listed in Table 1 were recorded in deionized water with a Jasco J-700 spectropolarimeter. A quartz cell of 1-cm optical path length was used, and the following setup was maintained: bandwidth, 1 nm; time constant, 2 s; scan rate, 20 nm/min. Four spectra corrected for background were averaged for each sample. For samples 1 and 5, a concentration of ∼3.0 × 10-3 monomol/L was selected, while for the other samples, a concentration of ∼1.5 × 10-3 monomol/L was used. Results and Discussion Relationships between Conformation and (Pattern of) Substitution for Galactose-Modified Alginate. Alginate from two different sources, namely, L. hyperborea and M. pyrifera, has been modified introducing galactose moieties on the carboxylic groups of the polymer. The galactosesubstituted polymers prepared, including the degree and pattern of substitution as determined by 1H NMR, are listed in Table 1. Because the synthetic procedure, as previously reported,26 was performed under acidic conditions (pH 4.5), the lack of degradation of the polymeric chain needs to be assessed. In this perspective, a high-performance sizeexclusion chromatography combined with refractive indexmultiple angle laser light scattering (HPSEC-RI-MALLS) analysis was performed on samples from 1 to 7. The weightaverage molecular weight (Mw) together with the z-average radius of gyration (〈sz2〉1/2) obtained are reported in Table 2. The increment in Mw observed for the modified polymers in comparison with that of the native samples is very well related to the degree of substitution. In fact, considering the Mw of the unsubstituted samples, there is a reasonable agreement (within a 10% error) between the Mw obtained by means of HPSEC-MALLS analysis and the one calculated on the basis of the degree of substitution. This tendency was confirmed for both alginate samples, evidencing on one hand that no degradation effect has taken place during the coupling of alginate with galactosylamine under acidic conditions and, on the other hand, that no aggregation has occurred. To determine the effect of the galactose introduction on the hydrodynamic properties of the polymeric chain, the intrinsic viscosity, [η], of samples from 1 to 7 was measured at 25 °C in NaCl 0.1 M. The results are reported in Table 3,
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Galactose-Substituted Alginate 2 Table 3. Intrinsic Viscosity of Native and Galactose-Subsituted Alginate Samples from L. hyperborea and M. pyrifera A. Effect of Galactose Substitution sample
ds (%)a
[η] (dL/g)b
k′c
k′′d
1 2 3 4 5 6 7
0 7 19 36 0 16 38
5.46 ( 0.05 3.98 ( 0.04 3.19 ( 0.06 3.64 ( 0.04 6.54 ( 0.06 3.83 ( 0.06 4.59 ( 0.08
0.47 0.43 0.54 0.48 0.52 0.53 0.48
0.11 0.11 0.06 0.10 0.10 0.08 0.11
B. Effect of Reduced Charge Density source
Re
(1 - R) × 100f
[η] (dL/g)b
k′c
k′′d
L. hyperborea L. hyperborea L. hyperborea L. hyperborea M. pyrifera M. pyrifera M. pyrifera
1 0.93 0.81 0.64 1 0.84 0.62
0 7 19 36 0 16 38
5.46 ( 0.05 5.32 ( 0.03 4.53 ( 0.03 3.93 ( 0.05 6.54 ( 0.05 6.29 ( 0.03 4.76 ( 0.03
0.47 0.41 0.48 0.46 0.52 0.56 0.62
0.11 0.12 0.10 0.11 0.10 0.08 0.04
a Total degree of substitution determined by 1H NMR. b Intrinsic viscosity reported as mean ( SE from values determined by means of eqs 1 and 2 (R2 ) correlation coefficient > 0.989 for all linear fits of eqs 1 and 2). c k′ represents the Huggins constant. d k′′ represents the Kraemer constant. e Degree of ionization (eq 4). f Degree of protonation.
section A. It is important to notice that all of the modified samples, from both L. hyperborea and M. pyrifera, showed a lower value of [η] when compared to the unsubstituted sample. Furthermore, for alginate from L. hyperborea, a monotonic decrease of the intrinsic viscosity was experienced when considering samples 1, 2, and 3 (i.e., with 0%, 7%, and 19% of galactose introduced, respectively), followed by a clear inversion of this tendency for sample 4 characterized by a higher degree of substitution (36%). The same trend of the intrinsic viscosity on the amount of galactose introduced was found analyzing the modified alginates from M. pyrifera. In the case of galactose-substituted alginate samples, it has already been pointed out that when the degree of substitution is higher, the molecular weight (Mw, Table 2) is higher. A decrease of the intrinsic viscosity should then be expected if compared with the unmodified polymer samples, under the assumption of an equal hydrodynamic volume.33 The value of the intrinsic viscosity of modified alginate samples should then be properly corrected for the increase of molecular weight determined by the introduction of galactose moieties. Such corrected value of [η], [η]gal estim, can be simply estimated by using eq 5: alg [η]gal estim ) [η]
Mds)0 Mds
(5)
[η]alg being the intrinsic viscosity of unmodified alginate, Mds)0 the molecular weight of the repeating unit of nonderivatized alginate, and Mds the molecular weight of the repeating unit of the galactose-substituted alginate (calculated on the basis of the degree of substitution). The difference gal gal ∆[η]gal ) [η]gal meas - [η]estim was calculated, [η]meas being the intrinsic viscosity measured for the different galactosesubstituted polymers (Table 3, section A). If the increase of the degree of substitution was accompanied by no major
change in the hydrodynamic value of alginate, the plot of ∆[η]gal vs ds should be zero (see dotted lines in Figure 1a,b). The experimental values of ∆[η]gal show a marked negative dependence on ds, for both samples (see square symbols in Figure 1a,b), pointing to the presence of additional effects. Obviously, the first one to check for is the well-established dependence of the value of [η] of a polyelectrolyte on the charge density along the chain.34 To assess the purely electrostatic effect of a reduced charge density on the unsubstituted polymer chain, the intrinsic viscosity of alginate from L. hyperborea with values of the degree of ionization, R, of 0.93, 0.81, and 0.64 (i.e., with a net charge density corresponding to that of samples 2, 3, and 4, respectively) was measured. Similarly, the [η] of alginate from M. pyrifera with R of 0.84 and 0.62 (corresponding to the net charge density of samples 6 and 7) has also been determined. As it was easily predictable, the values of [η] obtained, reported in Table 3, section B, show a monotonic decrease with decreasing the degree of ionization (i.e., decreasing the net charge density). Indeed, a correction for the modification in the molar mass of the repeating unit of the polymer should be applied also to the viscosity measured at different values of the degree of ionization (because of the replacement of Na+ with H+). Therefore, the theoretical value of the intrinsic viscosity at different R values, [η]Restim, has been estimated using eq 6: [η]Restim ) [η]alg
Mds)0 MR
(6)
[η]alg being the intrinsic viscosity of unmodified alginate at R ) 1 (sodium salt), Mds)0 the molecular weight of the repeating unit of alginate in the full sodium form, and MR the molecular weight of the repeating unit of alginate at different R values. The difference ∆[η]R ) [η]Rmeas [η]Restim was calculated, [η]Rmeas being the intrinsic viscosities measured for alginate at different R values (Table 3, section B), respectively. The dependence of these two values on the degree of substitution is reported in Figure 1a for L. hyperborea and in Figure 1b for M. pyrifera as circles. In both cases, the values of ∆[η]R are negative for all nonzero values of (1 - R), thus ruling out that the decrease in the intrinsic viscosity, reported in Table 3, cannot be explained only considering the variation in the molecular weight of the salt form of the repeating unit upon replacement of Na+ with H+. Rather, it must arise from the well-known decrease of the conformational dimensions of the polyelectrolyte chain upon reducing the net charge density. 34 Starting from alginate from L. hyperborea (Figure 1a), the trend of ∆[η]R is a clearly decreasing monotonic (sigmoidal) curve, as qualitatively expected. In contrast, ∆[η]gal is decreasing from sample 1 to 3 and increasing from sample 3 to 4. Furthermore, considering samples 2 and 3, their ∆[η]gal values are lower than those of unsubstituted alginate with the same degree of protonation (i.e., with an equal net charge on the polymer). This consideration leads to the conclusion that, in both samples 2 and 3, some other conformational effect reduces the expansion of the polymer chain, in addition to the expected electrostatic one. When
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Figure 1. Dependence of ∆[η]gal (9) and ∆[η]R (O) on the degree of galactose substitution and the degree of protonation, (1 - R) × 100, for alginate from (a) L. hyperborea and (b) M. pyrifera sources. Lines are drawn to guide the eye.
we look at the pattern of the galactose substitution (Table 1), sample 2 displays all of the substitution located on a G residue neighboring an M residue. Similarly, in the case of sample 3 a consistent part of the total amount of galactose is still introduced on G residues neighboring an M residue. This allows us to suggest that the introduction of galactose moieties on alternating sequences significantly increases the degrees of conformational freedom. Considering the alginate sample from L. hyperborea with a galactose content of 36% (sample 4), a sudden increase of ∆[η]gal, compared to sample 3, has been observed. Obviously, such behavior cannot be explained by taking into account the further decrease of the net charge; it rather shows that a higher hydrodynamic volume has taken place as a consequence of a re-extension of the polymer chain. In the case of sample 4, the galactose residues are introduced mainly on a G residue neighboring
a G residue, as reported in Table 1; when these residues along the chain are extensively substituted, the chain segments experience a hampered conformational freedom leading to an increased coil expansion, just the opposite of what is expected from a bare charge density reduction. The alginate chain takes a less-extended conformation when the substitution affects mainly G residues on alternating sequences, whereas a more-extended conformation is adopted when the substitution is mainly located on G diads. In the case of Figure 1b, that is, for alginate from M. pyrifera source, the same behavior as in Figure 1a has been found: a decrease of hydrodynamic properties for low degrees of substitution (mainly located on a G residue neighboring an M residue) followed by an increase of hydrodynamic dimensions when relatively high amounts of galactose are introduced on GG residues.
Galactose-Substituted Alginate 2
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Figure 3. Intrinsic viscosity as function of ionic strength for (9) sample 5, (b) sample 6, and (2) sample 7. Values reported as mean ( SE from intrinsic viscosity values measured according to eqs 1 and 2.
Figure 2. Dependence of the ratio 〈sz2〉3/2/Mw on the degree of galactose substitution for (a) L. hyperborea and (b) M. pyrifera. Lines are drawn to guide the eye.
The analysis of the rms value of the radius of gyration 〈sz2〉1/2 (see Table 2) led to a similar conclusion upon the effect of galactose substitution on chain expansion. In the alginate sample from L. hyperborea, a considerable decrease of the overall dimensions with respect to those of unsubstituted polymers occurs when introducing both 7% and 19% of galactose on the carboxylic groups. A similar behavior was shown by alginate from M. pyrifera, in which a 16% substitution causes a decrease of the radius of gyration compared to the unmodified material. In both cases, the modification of the carboxylic groups with sugar residues (up to 19%) causes a less-extended conformation of the polymer chain. At variance, when the amount of galactose introduced in the chain reaches 36% (sample 4) and 38% (sample 7) for L. hyperborea and M. pyrifera, respectively, an increase of the radius of gyration was observed with respect to samples 3 and 6, respectively. Figure 2 a,b represents the overall volume of the molecule (〈sz2〉3/2), normalized for the average molecular weight, Mw (to account for the increase of the molar mass when increasing the degree of substitution), as a function of the degree of substitution, ds. In both cases, that is, for alginate from L. hyperborea and M. pyrifera source, there is a very high similarity with the trend observed for the intrinsic viscosity, thus confirming the previous conclusions. The results produced so far clearly show that a complex change of conformation takes place upon substitution of
alginates with increasing amounts of galactose, depending upon the different sequences of monomeric components affected. Because a bare effect of the variation of charge density has been ruled out, it is reasonable to presume that the observed effect stems from a significant modification of the conformational space available to the affected uronic monomers, thereby producing a marked effect on the overall chain flexibility. A qualitative method to assess this hypothesis exploits the ionic strength dependence of the intrinsic viscosity of a polyelectrolyte. The B-parameter was related by Smidsrød31 to chain stiffness; considering the slope S of the plot of [η] against I-1/2 (with I ) ionic strength) (Figure 3), the following relation stands (eq 7): S ) B[η]0.1ν
(7)
[η]0.1 being the intrinsic viscosity at 0.1 M NaCl and ν ) 1.3. More rigid backbones show lower values of B, whereas more flexible chains have a marked dependence of [η] on I (typically the value of the parameter B ranges from 5.25 × 10-3 for a very stiff molecule such as xanthan35 to 0.2 for carboxymethylamylose31). In the case of sample 5, a value of B ) 0.052 was obtained, describing alginate from M. pyrifera as a semiflexible coil. It has been reported36,37 that the B does not depend on the degree of dissociation or of substitution of a polycarboxylate31 such as alginate. In the case of sample 6, the B value was found to be 0.067, significantly higher than that of the unsubstituted polymer, evidencing an intrinsically more flexible conformation. Interestingly enough, for sample 7 a value of B equal to 0.057 was obtained, that is, close to that of sample 5, evidencing that the highly modified polymer shows a stiffness closer to unmodified sample 5 than to 6. Evaluation of the Persistence Length of the GalactoseSubstituted Alginate. The qualitative indications on the chain stiffness of native and modified alginate given by the B-parameter analysis can find a more sound quantitative verification by use of two different methods to calculate the persistence length38 of samples from 1 to 7.
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Table 4. Values of Persistence Length (q), Weight-Average Degree of Polimerization (Nw), Weight-Average Contour Length (Lcw), Weight-Average of the Number of Kuhn segments (NK), and Φ A. Comparison between Present Alginate Samples and Values Previously Reported in the Literature sample
source
FG; FGG
solventb
q (nm)
Nwc
Lcwd (nm)
z
N Ke
〈sz2〉/(2q)2
Φ × 1023 (mol-1)
1 AA1a 5 AA2a
L. hyperborea L. hyperborea M. pyrifera M. pyrifera
0.69; 0.56 0.72; 0.58 0.42; 0.20 0.42; 0.22
I II I II
23.9 25.6 15.2 14.5
583 1005 996 972
269 463 486 470
1.31 1.61 0.99 1.04
5.63 9.04 15.67 16.19
1.42 1.33 4.87 2.97
0.47 0.42 0.83 0.74
B. Native and Galactose-Substituted Alginate from L. hyperborea sample
ds (%)
q (nm)f
Nwc
Lcw (nm)d
z
NKe
〈sz2〉/(2q)2
Φ × 1023 (mol-1)g
1 2 3 4
0 7 19 36
23.9 18.7 9.2 10.6
583 592 573 566
269 273 264 261
1.31 1.11 1.21 1.06
5.63 7.30 14.36 12.31
1.42 1.83 5.64 4.35
0.47 0.53 1.26 1.24
sample
ds (%)
(nm)f
5 6 7
0 16 38
C. Native and Galactose-Substituted Alginate from M. pyrifera
q
15.2 7.7 10.9
Nwc
Lcw (nm)d
z
NKe
〈sz2〉/(2q)2
Φ × 1023 (mol-1)g
996 1104 1110
486 533 536
0.99 0.76 1.03
15.67 34.64 24.61
4.87 13.60 7.80
0.83 1.53 1.26
a Data reported in ref 42. b I ) NaCl 0.1 M; II ) Na SO 0.05 M, EDTA 0.01 M (pH ) 6). c N ) M /M with M ) molecular weight of the repeating 2 4 w w 0 0 unit of the galactose-substituted alginate. d Lcw ) lNw. e NK ) (Lcw/(2q)). f Persistence length calculated on the basis of eq 8 using data from SEC-MALLS g (solvent Na2SO4 0.05 M, EDTA 0.01 M (pH ) 6)). Value calculated using eq 12 ([η] in mL/g determined in NaCl 0.1 M, q and l in cm).
In the first case, the Doty-Benoit equation (eq 8),39 obtained for wormlike chains in unperturbed conditions, was used.
{( )
( )[ ( )]}
l 2q q 〈sz2〉 - q2 Nz -1+ 13q Nwl Nnl
)0
(8)
where q is the persistence length and Nn, Nw, and Nz are the number-average, weight-average, and z-average degree of polymerization, respectively. To calculate q, Mn, Mw, and Mz values have been obtained for each sample using HPSECRI-MALLS, while the molecular weight of the repeating unit was calculated on the basis of the degree of substitution obtained with 1H NMR. In this case, the virtual bond length, l, was assumed to be linearly dependent on the composition, in terms of G and M content, of the two different alginate samples, as often done in similar calculations on heteropolysaccharides.40-42 Therefore, starting from the values obtained from X-ray fiber diffraction data,43 that is, lM ) 5.17 Å for M and lG ) 4.35 Å for G residues, we have assumed an average value of virtual bond length of 4.61 and 4.83 Å for alginate samples from L. hyperborea and M. pyrifera, respectively. Table 4, section A reports the calculated persistence length values as obtained by use of eq 8 for samples 1 (from L. hyperborea, 23.9 nm) and 5 (from M. pyrifera, 15.2 nm). Table 4, section A reports also the values of persistence length of samples of very similar composition obtained by using the Bohdanecky equation.42 The two sets of data are in very good agreement. Furthermore, it is important to notice that the different ionic composition of the aqueous solvent used in the present work (as compared to ref 42) does not lead to a significant variation in the value of the persistence length. (Persistence length values for alginate reported in the literature as high as >50 nm32 or as low as
