Elsevier

Carbohydrate Polymers

Volume 148, 5 September 2016, Pages 52-60
Carbohydrate Polymers

Single molecule investigation of the onset and minimum size of the calcium-mediated junction zone in alginate

https://doi.org/10.1016/j.carbpol.2016.04.043Get rights and content

Highlights

  • Ca2+-mediated crosslinking of oligoguluronic acids (“oligoGs”) with dp 6–20 was studied by single molecule force spectroscopy.

  • The minimum length of an oligoG required to form a stable eggbox junction zone is 8 monomers.

  • Rapidly forming weak Ca2+-mediated bonds initially outcompete and hinder the slower formation of stable egg box junctions.

  • This competition between crosslinking modes accounts for the long relaxation times seen during the formation of alginate gels.

Abstract

One of the principal roles of alginate, both natively and in commercial applications, is gelation via Ca2+-mediated crosslinks between blocks of guluronic acid. In this work, single molecule measurements were carried out between well-characterised series of nearly monodisperse guluronic acid blocks (‘oligoGs’) using dynamic force spectroscopy. The measurements provide evidence that for interaction times on the order of tens of milliseconds the maximum crosslink strength is achieved by pairs of oligoGs long enough to allow the coordination of 4 Ca2+ ions, with both shorter and longer oligomers forming weaker links. Extending the interaction time from tens to hundreds of milliseconds allows longer oligoGs to achieve much stronger crosslinks but does not change the strength of individual links between shorter oligoGs. These results are considered in light of extant models for the onset of cooperative crosslinking in polyelectrolytes and an anisotropic distribution of oligoGs on interacting surfaces and provide a timescale for the formation and relaxation of alginate gels at the single crosslink level.

Introduction

Alginate is the collective term for a group of binary copolymeric polysaccharides synthesised by some algae and bacteria (Draget, Smidsrød, & Skjåk-Bræk, 2005; Gorin & Spencer, 1966; Linker & Jones, 1966). The two monomers comprising the copolymer are α-l-guluronic acid (‘G’) and β-d-mannuronic acid (‘M’). Since M and G are epimers of each other, the alginate polymer is synthesised as polymannuronic acid and epimerisation of individual residues within the polymannuronic acid chain to guluronic acids is carried out by a family of epimerases (Haug and Larsen, 1971a, Haug and Larsen, 1971b, Larsen and Haug, 1971). This enzymatic epimerisation results in varied patterns of the sequences GMG and GG being generated within the polymannuronic acid, with different organisms having different epimerases that make alginates specific to each species (Campa, Holtan et al., 2004; Campa, Oust et al., 2004; Hartmann, Holm, Johansen, Skjåk-Bræk, & Stokke, 2002; Høidal, Ertesvåg, Skjåk-Bræk, Stokke, & Valla, 1999; Holtan, Zhang, Strand, & Skjåk-Bræk, 2006).

The key function of alginates, both in vivo and as exploited commercially, is their gelling ability (Draget, Skjåk-Bræk, & Smidsrød, 1997; Draget & Taylor, 2011). The chief mechanism of gelation in alginates is through the metal cation (typically Ca2+)-mediated formation of so-called ‘eggbox’ junction zone crosslinks between sequences of Gs in interacting polymers of alginate (Grant, Morris, Rees, Smith, & Thom, 1973): multiple pairs of neighbouring G residues on opposing alginate chains are coordinated by Ca2+ ions that fit into the pockets created by the G pairs, resembling eggs in an eggbox (see Fig. 1 for an illustration). More recent evidence for the eggbox junction zone configuration comes from detailed X-ray diffraction studies (Sikorski, Mo, Skjåk-Bræk, & Stokke, 2007), and its importance as a gelling mechanism is borne out by the finding that alginates comprising long uninterrupted sequences of polyG form the strongest gels in the presence of divalent cations (Skjåk-Braek, Smidsrod, & Larsen, 1986). More recently still, molecular dynamic and Monte Carlo simulations (Plazinski, 2011; Stewart, Grey, Vasiljevic, & Orbell, 2014) have revealed alternative Ca2+-dependent modes of crosslinking two or more polyG strands, including variants of the eggbox junction as well as a perpendicular junction involving two polyG strands and three Ca2+ ions, while a similar ‘tilted eggbox’ crosslink has been proposed independently (Borgogna, Skjåk-Bræk, Paoletti, & Donati, 2013) to reflect the intial stages of crosslinking between two alginate strands. The minimum length of an oligoguluronate (oligoG) necessary to form a strong eggbox junction zone has been considered to be eight residues (allowing for four Ca2+ ions to be crosslinked) based upon a cooperative model for junction formation (Stokke, Smidsrød, Bruheim, & Skjåk-Bræk, 1991) and corroborated by leaching studies that showed only polymers with polyG sequences shorter than eight were leached from Ca-alginate gels (Stokke, Smidsrød, Zanetti, Strand, & Skjåk-Bræk, 1993). A detailed analysis of the dependence of the strength of the Ca2+-mediated interaction between oligoGs on the length of oligoG sequences, however, has not been carried out before, due in part to the lack of well-characterised oligoGs.

We set out to establish the minimum length of oligoG required to form a stable eggbox junction zone and to characterise the onset of eggbox formation using single molecule force spectroscopy measurements conducted by atomic force microscopy (AFM). AFM has previously been used to investigate the binding of the epimerase AlgE4 to its alginate substrate, to study the effect of oligoGs on reducing the interactions between alginate and mucins as well as in an attempt to characterise the relative proportions of M and G in alginates based upon the presence or absence of the chair-boat transition (Sletmoen, Maurstad, Nordgård, Draget, & Stokke 2012; Sletmoen, Skjåk-Bræk, & Stokke, 2004; Williams, Marshall, Haverkamp, & Draget, 2008). The availability of such a single molecule measurement method, in conjunction with a series of nearly monodisperse oligoguluronic acids, allows us to explicitly measure for the first time the strength of interaction between oligosaccharide pairs in an eggbox junction.

Section snippets

Preparation of purified oligoGs

OligoGs were fractionated from partially hydrolysed polyG by size exclusion chromatography and freeze dried as previously described (Ballance et al., 2005). Size was assessed with HPAEC-PAD and compositional purity F(G) and degree of polymerisation (DP(n)) were calculated according to both of the methods described in a previous work (Grasdalen, Larsen, & Smidsrød, 1979) from 1H NMR spectra recorded on a Bruker Avance 400 MHz spectrometer (Campa, Holtan et al., 2004, Campa, Oust et al., 2004):

AFM of interactions between oligoGs

OligoGs with lengths from 6 to 20 monomers were coupled to short PEG spacers and attached to both an AFM probe and a mica surface and force curves were collected in the absence and presence of 2 mM CaCl2 before addition of 20 mM EDTA. Because the stoichiometry of the GG-Ca2+-GG eggbox junction zone complex is 2:1:2 (see Fig. 1), the series of oligoGs used can form complexes between pairs of oligoGs by complexation with approximately 2, 3, 4, 6 and up to 10 Ca2+ ions (Table 1). The PEG-coupling

Conclusion

The aims of this work were to establish, at the single molecule level, the minimum sequence of pairs of guluronic acids necessary to induce a strong junction zone in the presence of Ca2+, and thence to characterise the onset of this minimum junction zone and follow its evolution to longer junctions. We have shown experimentally for the first time that pairs of at least 8 guluronic acids are required to form a strong, stable Ca2+-mediated junction zone. The apparent reduction in strength of

Acknowledgements

The authors would like to acknowledge funding from Biotechnology and Biological Sciences Research Council (UK) grant H019294 (KB and AR); Norwegian Research Council grant MARPOL 10 399 200 (OA and GS-B) and Science Without Borders (MN).

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