Acacia senegal Gum:  Continuum of Molecular Species Differing by Their Protein to Sugar Ratio, Molecular Weight, and Charges

The main chemical and physical features of the Acacia senegal exudate gum and its molecular fractions isolated by chromatographies were determined using a wide variety of methods. Three main molecular fractions were isolated after hydrophobic interaction chromatography (HIC) and biochemical analyses confirmed the presence of an arabinogalactan-peptide (FI), an arabinogalactan-protein (FII), and a glycoprotein (FIII) fraction as described commonly in the literature. Further purification of FIII using size exclusion chromatography revealed three distinct populations. A wide molecular weight distribution within each population with the presence of at least two distinct molecular species per population was identified by high performance size exclusion chromatography coupled to on line multi-angle laser light scattering (HPSEC-MALLS). In addition, both sugars content (neutral and uronic acids) and UV profiles revealed that FIII was composed of a continuum of molecular species differing both by their protein-to-sugar ratio and molecular weight. FI and FII had average molecular weight M(w) of 2.86 x 10(5) and 1.86 x 10(6) g.mol(-1), respectively, and a low polydispersity index (M(w)()/M(n) approximately 1.3). The three populations identified in FIII after HIC separation had M(w) of 2.67 x 10(6), 7.76 x 10(5), and 2.95 x 10(5) g.mol(-1) and very low polydispersity indexes (1.13, 1.04, and 1.01). Estimation of the polypeptide backbone length in the three fractions gave 43, 2253, and 4443 amino acid residues, respectively, hydroxyproline (Hyp) and serine being the most prominent residues within FI and FII, Hyp and Asx (asparagine + aspartic acid) within FIII. Secondary structure prediction from circular dichroism data resulted in polyproline II, beta-sheet, and random coil structures for FII and FIII, whereas no secondary structure was identified in FI. The existence of exposed tryptophanyl residues to the solvent was noticed by fluorescence in FII and FIII, tryptophan residues being absent from FI. In addition, 8-5' non cyclic diferulic acid was identified to be covalently linked to carbohydrate moieties of FII. Infrared spectroscopy identified the different vibrations of saccharidic and peptidic bonds with absorbance amplitudes in agreement with sugar and protein elementary analyses. Titration measurements in order to evaluate the number of charges on total Acacia gum and its molecular fractions revealed that 100% of charges came from polysaccharidic moieties (i.e., glucuronic acids) in FI. Charges coming from polysaccharidic moieties were of 91.3% and 37.9% for FII and FIII, respectively, the remaining 8.7% and 62.1% charges in FII and FIII molecular fractions coming from the polypeptidic backbone.