Analysis of pectate lyase-generated oligogalacturonic acids by high-performance anion-exchange chromatography with pulsed amperometric detection

Abstract

Traditionally, UV absorption detection ( A 235) of 4,5-unsaturated bonds has been used to evaluate the depolymerization of pectic polysaccharides by pectate lyase (PL). This approach ignores the generation of oligogalacturonic acids without a 4,5-unsaturated function that appear early in the time course of PL cleavage. We found that high-performance anion-exchange chromatography and pulsed amperometric detection (HPAEC-PAD) could separate and detect oligogalacturonic acids both with and without 4,5-unsaturated functions at their nonreducing ends in PL digests of polygalacturonic acid (PGA) and tobacco cell walls. The “recombinant” PL used was free of endopolygalacturonase activity. Peaks were identified in PL digest chromatograms by retention time agreement with oligoglacturonic acid standards purified by preparative high-performance liquid chromatography and a PGA autoclave hydrolysate that contained oligogalacturonic acids up to degree of polymerization (dp) 40, which lacked the 4,5-unsaturated function. When comparing like dp oligogalacturonic acids, those without the 4,5-unsaturated function eluted earlier from the HPAEC CarboPac PA1 column than did their unsaturated counterparts. These techniques provide the first means for the dp assignment of oligogalacturonic acids generated by both hydrolytic and lytic cleavage of polygalacturonic acid and plant cell walls.