Hydrophobic Interactions of Succinoglycan Dimers Isolated from Sinorhizobium meliloti with Hydrophobic Fluorescence Probes, 8-anilino-1-naphthalenesulfonate and 6-p-toluidino-2-naphthalenesulfonate

Abstract

Succinoglycan, an acidic exopolysaccharide, is produced by the nitrogen-fixing bacterium, Shinorhizobium meliloti. The main chain consists of a β-1,3, β-1,4, and β-1,6 linked octasaccharide subunit containing one galactose at the reducing end and seven glucose residues. Also, the repeating unit carries pyruvyl, acetyl, and succinyl modifications. The acetyl group is located at the C6 position of the third sugar residue from the reducing galactose, the succinyl group is located at the C6 position of the seventh sugar, and the pyruvyl group is linked to the eighth sugar residue through a 4,6-ketal linkage. The succinoglycan has high molecular weight (HMW) forms consisting of hundreds of octasaccharide units and low molecular weight (LMW) forms that are composed of monomers, dimers, and trimers of the octasaccharide unit. The LMW succinoglycan offers the advantages of the HMW succinoglycan for research because it is not viscous and the structure is simpler than the HMW succinoglycan. Also, the LMW succinoglycan is of particular interest, as some reports have described that it is able to restore the ability of invasion-deficient S. meliloti mutants to invade nodules. In addition to investigation of its biological activity, physico-chemical properties of the LMW succinoglycan have been studied. Monomers of succinoglycan have been isolated and investigated as chiral additives in capillary electrophoresis (CE) for the chiral separation of some flavonoids. The effect of enantioseparation is known to be dependent on the succinate moiety of the monomer structure. Also, flavonoid enantioseparations using succinoglycan monomer show that the monomer may directly interact with flavonoids produced by the host plant. However, there have been no reports on physico-chemical properties of dimers or trimers. Herein, we examine the structural properties of the dimers. The chemical structures of succinoglycan dimers are also shown in Figure 1(a). Fluorescent probes are defined as small molecules that undergo changes in one or more of their fluorescent properties as a result of hydrophobic interaction with environmental macromolecules. As representative fluorescent probes, 8-anilino-1-naphthalene sulfonate (ANS) and 6-ptoluidino-2-naphthalenesulfonate (TNS) have been used to explore the hydrophobicity of biomolecules. The chemical structures of both fluorescent probes are shown in Figure 1(b) and 1(c). In the present study, the fluorescence of ANS and TNS, respectively, in an aqueous solution was investigated under the presence of succinoglycan dimers (D1, D2, D3, and D4), and the hydrophobic character of the dimers was analyzed. Based on double reciprocal plots of the fluorescence increments depending on the dimer concentration, the association constants could be estimated. The results indicate that association constants of ANS and TNS, respectively, with D2 are the largest among the 4 types of