Analysis of pectin structure part 3 - Quantification of the amount of galacturonic acid residues in blocksequences in pectin homogalacturonan by enzymatic fingerprinting with exo- and endo-polygalacturonase II from Aspergillus niger

A method to determine the amount of galacturonic acid in blocksequence (BS) in pectin homogalacturonan (HG) is described. The method is based on a com bination of endopolygalacturonase II (endo-PG II) and exopolygalacturonase (exo-PG) digestion followed by quantification of the liberated galacturonic acid monomer. The amount of monomers released is directly related to the a mount of non-esterified galacturonic acid units located between two other n on-esterified galacturonic acids units on the HG chain. The amount released for exo-PG digestion only corresponds to the BS located at the non-reducin g end of the polymer. The difference between total- and exo-BS was calculat ed to be the amount of endo-BS located either within or on the reducing end of the HG. Three series of model pectins obtained by de-esterification of a high-eater pectin with either plant pectin methyl-esterase (p-PME, P-seri es), fungal pectin methyl-esterase (f-PME, F-series) and chemical de-esteri fication using base (B-series) were analysed and compared with a fully de-e sterified pectic acid sample obtained from the same raw material. Clear dif ferences for the increase of the amounts of blocksequence could be seen bet ween de-esterification of the P- and F-series samples supporting a blockwis e and a homogenous de-esterification mechanism, respectively. f-PME and bas e treatment showed only minor differences in the increase of galacturonic a cid units in BS, despite differences seen in their methyl-esterification pa ttern. Differences between the amounts of galacturonic acid located in exo- and endo-BS, provided evidence for the need of a certain start side or blo cklength for p-PME to de-esterify blockwise. (C) 2000 Elsevier Science Ltd. All rights reserved.