Partial Purification of Peroxidase lsozymes with Altered Substrate Specificity from Flax stem Cell Walls

Summary A comparison of the peroxidase activities of ionically and covalently bound but Driselase-solubilized cell-wall proteins from flax stems showed that the Driselase-solubilized proteins had a higher relative ability to oxidize coniferyl alcohol (CA) in the presence of hydrogen peroxide than the ionically bound proteins. In fact, when the ratio of peroxidase activity against CA to activity against o -dianisidine ( o -D) was calculated, the Driselase-solubilized wall proteins appeared to have four times the affinity for the oxidation of CA than the ionically bound proteins. To test if this phenomenon extended to individual isozymes, the most abundant, also the most anionic, isozyme was purified from each fraction by ion-exchange chromatography on DEAE-Sepharose followed by affinity chromatography on Concanavalin-A Sepharose. The peroxidase isozymes from the Driselase-solubilized and the ionically bound fractions (D4 and C4 resp.) had identical elution properties in both steps. After ion-exchange chromatography, which separated isozymes C4 and D4 from other isozymes, isozyme D4 had a CA/ o -D ratio that was 1.8-fold higher than that of isozyme C4. Affinity chromatography on Concanavalin-A Sepharose increased the specific activity of isozyme D4 by 37-fold when measured with o -D but by only 4.8-fold when measured against CA. This disparity suggests that activity against CA has been lost from the main peroxidase peak. A protein peak with a lower affinity for Concanavalin-A contained appreciable peroxidase activity against CA but extremely low activity against o -D. This protein may be an artefactual form generated during glycanase solubilization of covalently bound enzymes from the cell wall. This peroxidase appears to be a partially deglycosylated form of isozyme D4 that has an altered substrate specificity.