Structural characterization of arabinan from Pinus radiata cambium tissue : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University, Palmerston North, New Zealand
Cambium tissue isolated from Pinus radiata was frozen, thawed, washed with cold phosphate buffer, then extracted with phosphate buffer by passage through a French pressure cell. The washings and French press extract were dialyzed. The pectic components partially purified by chromatography on DEAE-cellulose. These polymers bound weakly to DEAE-cellulose at pH 8.0. In both extracts, an early acidic peak of high arabinose and galactose content was separated from a late acidic peak with mostly uronic acid. Within the early acidic peak, successive fractions showed an increase in uronic acid and rhamnose content. Treatment of the extracts with boiling water caused a decrease in binding of the arabinose-rich polymer to the DEAE cellulose reflected by earlier elution, and a minor decrease in average molecular size, consistent with β-elimination of the pectic backbone. The acidic fractions were then further fractionated by gel filtration on Superose and Superdex FPLC columns. The earliest of these acidic fractions had ratios of Ara:Gal:Rha = 30:16:1, this suggesting relatively long side chains of about 46 residues, and were mostly excluded by the Superdex-peptide column. The native molecular mass of the arabinose-rich polymers was determined by size exclusion chromatography and molecular masses of extract A and B were observed to be 40-100 kDa and up to >500 kDa, respectively by Superose and Superdex FPLC columns. Other characterization studies on the arabinan involved quantitative analysis of the monosaccharides derived by sulfuric acid hydrolysis and quantitation of the sugar linkages using methylation analysis. Methylation analysis suggested a highly branched arabinan structure and the presence of terminal galactosyl residues on a branched arabinan core. From the results, a tentative structure has been suggested for the arabinose rich polymers. Arabinose was mainly present as 3-, 5-, 3,5-, and terminal linked residues. Galactose was mainly present as terminal residues.