Isolation and Characterization of Rhamnogalacturonan Oligomers, Liberated During Degradation of Pectic Hairy Regions by Rhamnogalacturonase

Digests of modified hairy regions of apple pectin (MHR) obtained after degradation by rhamnogalacturonase (RGase) were analyzed for oligomer composition using high-performance anion-exchange chromatography and pulsed amperometric detection. A series of oligomers which appear to be characteristic of RGase degradation could be recognized. These oligomers were isolated on a preparative scale by size-exclusion chromatography and preparative anion-exchange chromatography and analyzed for sugar composition. 1H NMR spectroscopy showed that the oligomers consisted of between 4 and 9 sugar units with a backbone of alternating rhamnose and galacturonic acid residues, partly substituted with galactose residues linked to C-4 of the rhamnose moiety. The HPLC elution pattern showed that higher oligomers were also formed during incubation with RGase. These have the same basic structure but may contain other sugar units in addition to those given above. The oligomer composition of RGase digests of MHR isolated from apple, pear, leek, onion, carrot, and potato was very similar. Using anion-exchange chromatography to monitor the degradation of MHR at increasing incubation times, it was found that all the oligomers were present from the initial stages of the enzyme reaction and that the ratio between the different oligomers remained constant with time. Implications of these results for the structure of MHR and the mechanism of RGase action are discussed.