A new approach for studying interaction of the polygalacturonase-inhibiting proteins with pectins.

A method for determination of the interaction between pectins and proteins was developed using cross-linked polygalacturonic acid (CLPG) as the pectic substrate and polygalacturonase-inhibiting proteins (PGIPs). Defined water-insoluble pectins were prepared by chemical substitutions with acetyl or methoxyl groups on CLPG. In the presence of 0.1 M NaCl, PGIPs fully bound to CLPG but not to cross-linked alginic acid (CLAL), which had a similar pK(a) to CLPG, suggesting that the inhibitor was not simply bound to the substrate by nonspecific electrostatic interaction. Optimum binding of PGIPs to CLPG occurred at pH 2.4 to 4.7. The binding ability of the inhibitor to CLPGs with degree of methylation (DM) of 66% or degree of acetylation (DAc) of 133% was not significantly changed. In contrast, the DM of 82% or 95% decreased the binding. These results indicated that the carboxylic groups of galacturonic acid residues were involved in the recognition of the substrate by PGIPs.