Fine Structure Measurement

While in general the elucidation of biopolymer sequences has made great progress in recent years, polysaccharide fine structure determination is still not trivial, even in principle.This is largely owing to the fact that by the time a sample reaches the analysis lab it has been extracted from a functional cell wall and as such different chains will likely have had their fine structures modified dependent on their exact location. The tools of molecular biology such as PCR, that have so facilitated sequencing in nucleotides and proteins, are simply not available for polysaccharides and it cannot be taken for granted that there exist multiple copies of the same chain. Rather the sample is an intermolecular distribution of intramolecular sequences.

Pectin Work

Pectin is a complex carbohydrate polymer that plays an instrumental role in regulating the mechanical properties of the plant cell wall and has also found great utility in many diverse areas of science and technology. While the detailed structure of the pectin macromolecular assembly in-vivo is still a matter of debate (Vincken et al., 2003) most commercially available pectin samples can be considered as a collection of polymer chains each consisting of extended regions of homogalacturonan interspersed sparsely with regions of rhamnogalacturonan I. Even at this level of description complexity and heterogeneity abound. In particular, the distribution of methylesterification both among chains and along individual polymer backbones is a key determinant of molecular functionality. Indeed, cell wall enzymes routinely tailor DM distributions in order to exploit structure- function relationships based on the dependence of molecular association on the pattern of methylesterification (Willats et al., 2001). The measurement of such distributions is then vital in order to understand the role that fine structure modifications play in determining the functionality of pectin both in-vivo and in-vitro . It is envisaged that ultimately useful pectin structure-function models will not just incorporate information on DM distributions, but will require it in order to correctly predict the properties of pectin samples.

Intermolecular Degree of Methylesterification

Capillary electrophoresis has recently been shown to be a useful tool for the investigation of pectin methylester distributions. Most simply it can be used in order to measure the average DM of a pectin sample, since there is a linear relationship between the electrophoretic mobility and the average charge per residue. While many other methods perform this sample averaged DM measurement equally well, an advantage of the electrophoretic method is its inherent separation quality. For chains with lengths in excess of around 15 residues a symmetrical scaling of charge and hydrodynamic friction coefficient with the degree of polymerisation (DP) is found. This means that larger polymeric chains, regardless of their DP, elute according to their average charge density and, therefore, that each CE migration time marks species with a unique DM. Peak shapes thus reflect the intermolecular methylesterification distribution (the DM distribution among chains) of the sample.

While the co-injection and subsequent separation of pectin mixtures has provided strong evidence that the CE peak width is indeed a reasonable reflection of the intermolecular DM distribution, collecting and re-injecting fractions from the leading and following edges of a single peak has not been possible owing to the small sample volume that can be collected from a single CE run. However, in the same spirit, we have taken pectin fractions homogenous in molar mass, and subsequently fractionated these samples again, now on the grounds of charge (using IEC). The intermolecular methylester distribution of pectin samples fractionated from a mother sample (a) on the grounds of molecular weight and (b) on the grounds of charge.

It is worth noting that the validity of such a methodology for obtaining intermolecular DM information from CE hinges crucially on two major assumptions. Firstly, that contributions to peak widths arising from chromatographic factors are small compared to the breadth of the intermolecular mobility (charge) distribution, and secondly that the mobility is not significantly dependent on the intramolecular distribution of charge (methylesterification), so that the migration behaviour is determined simply according to the chain-averaged charge density. The first point has been largely addressed by observing the invariance of peak widths to changes in injection time, monitoring the co-injection and subsequent resolution of discrete samples, and performing calculations of the relative contributions expected from the relevant band-broadening mechanisms . The second point has been addressed by studying commercial pectin samples fractionated according to their calcium sensitivity and, more recently, homemade samples molecularly engineered to have a random or block wise DM distribution by exploiting demethylesterification using chemical saponification in contrast to processive enzymes. It was concluded that there was no statistically significant difference between the electrophoretic mobilities of pectins of equivalent DM with differing intramolecular arrangements of methylesterification.

Intramolecular Degree of Methylesterification

In general there are two approaches to the determination of the intramolecular distribution of methylesterification (the spatial distribution of methylesterified residues along the pectic backbone). These are i) to attempt to measure the distribution directly by recording a property of the residues that is sensitive to the local residue type environment, and ii) to fragment the chain according to rules that depend upon the residue type environment, and analyse the fragments generated. Broadly speaking NMR methods have been applied that follow the first methodology, while many enzymatic and chemical methods have been described that utilise the second approach.

Direct NMR Methodologies

We are currently using our excellent NMR facilities in order to develop methods for pectin fine structure elucidation, in particular examining the measurement of diad and triad frequencies of the methylester distributions.

Fragmentation Approach

Recent progress in this area, specifically addressing the intra-molecular distribution of methyl-esterified residues in pectic substrates, has been made using a fragmentation approach in which endo-polygalacturonase (endo-PG) is used to digest the polysaccharide and the subsequent (methyl-ester sequence dependent) digest pattern is determined. Hence, the separation, detection and quantification of partially methylated oligogalacturonide digest fragments play a key role in the elucidation of the fine structure of pectin. The structural characterisation of the partially methylesterified oligogalacturonide products is experimentally feasible for degrees of polymerisation (DP) below around 10. While the bulk of such work to date has been carried out using anion exchange chromatography and mass spectrometry, electrophoretic methods have also recently been reported as additional tools in pectic oligosaccharide analysis. These include the use of gel electrophoresis (PACE) coupled with fluorescent labeling, and capillary electrophoresis (CE) using UV detection of the unadulterated oligomers.

Oligogalacturonides

Detection, Separation and Quantification

Work in collaboration with Paul Dupree and Florence Goubet has been carried out in order to compare the information obtainable on partially methylesterified oligogalacturonides by PACE and CE.

Digest Simulation

We are also interested in exactly how much information the fragments of particular pectin digests contain about the pre-digested substrate. This will obviously depend in some way on the "fragmentation rules". It is interesting to consider two contemporary differing views from the literature about how well such rules are understood.

“ However, for a correct interpretation of the results, sufficient knowledge of the enzymes mode of action as well as detailed information on the methyl ester and GalA content of the degradation products is essential. Only when both conditions are met, can information be obtained that will allow for a reliable reconstruction of methyl ester distribution of the starting material. Pectin-degrading enzymes have been studied for several decades, providing adequate information on their mode of action and other more general characteristics. Especially the active site of the homogalacturonase (endo-PG) has been studied in detail .”

“Despite the large number of polygalacturonase-encoding gene sequences available in databases, very few studies have been designed to investigate the mode of action of the corresponding enzymes by analysing their subsite characteristics......therefore detailed knowledge about subsite architecture of these industrially important enzymes is scarce .”

We are working on producing viable in-silico models of enzymatic digestion in order to create a tangible link between polymeric fine structure and digest pattern.