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A new view of pectin structure revealed by acid hydrolysis and atomic force microscopy
Carbohydrate Research 345 (2010) 487–497
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A new view of pectin structure revealed by acid hydrolysis and atomic force microscopy Andrew N. Round a,b,*, Neil M. Rigby b, Alistair J. MacDougall b, , Victor J. Morris b a b
School of Pharmacy, University of East Anglia, Norwich NR4 7TJ, UK Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, UK
a r t i c l e
i n f o
Article history: Received 3 September 2009 Received in revised form 11 December 2009 Accepted 18 December 2009 Available online 28 December 2009 Keywords: Pectin Polysaccharide Structure Kinetics Microscopy AFM
a b s t r a c t Individual pectin polymers and complexes, isolated from the pericarp of unripe tomato (Lycopersicon esculentum var. Rutgers), were subjected to a mild acid hydrolysis and visualised and characterised by atomic force microscopy (AFM). The AFM images confirm earlier studies showing that individual pectic polysaccharides often possess long branches. The AFM data have been used to construct size and molecular weight distributions for the single molecules and complexes, from which the calculated numberaverage and weight-average molecular weights can then be compared directly with the published literature data on the rheology of bulk samples. Loss of the neutral sugars arabinose, galactose and rhamnose from the pectin samples does not significantly alter either the size or the branching density of the individual polymers, but is reflected in a breakdown of the complexes. Significant loss of galacturonic acid at long hydrolysis times was found to be accompanied by changes in the size and branching of the single polymers and further breakdown of the complexes. The results suggest that rhamnose, arabinose and galactose are not the major components of the individual polymers but are, instead, confined to the complexes. The polysaccharides represent a previously unrecognised branched homogalacturonan with a minimum mean size some three times larger than that previously reported. The complexes consist of homogalacturonans (HGs) held together by rhamnogalacturonan I (RG-I) regions. Comparison of the rate of depolymerisation of the homogalacturonans and complexes with the published data on changes in the intrinsic viscosity of bulk pectin samples, subjected to similar acid hydrolysis, suggests that the different rates of depolymerisation of RG-I and HG contribute separately to the observed changes in intrinsic viscosity during acid hydrolysis. Thus data obtained using a single molecule microscopy technique provides new insights into the behaviour in the bulk. Crown Copyright Ó 2010 Published by Elsevier Ltd. All rights reserved.
1. Introduction Pectins are complex biopolymers with as yet not fully understood functions within the plant cell wall.1 Pectic polysaccharides of non-graminaceous plants are a vital structural component of the plant cell wall and pectin extracts are commercially important to the food and pharmaceutical industries; in a normal western diet around 4–5 g of pectin is consumed each day. Pectin is used in a range of processed food products as a gelling agent or as a stabiliser in acidic milk drinks. The structure of pectin varies according to the source species, original tissue type and location, developmental or metabolic stage, environmental state and conditions of extraction. This complexity and, in particular, variability of structure undoubtedly hold the key to their role(s) in the cell wall, but also make detailed analysis of pectic polysaccharides a chal* Corresponding author. Tel.: +44 (0)1603 592985; fax: +44 (0)1603 592015. E-mail address: [email protected] (A.N. Round). Present address: National Starch LLC, Bridgewater, New Jersey, NJ 08807, USA.
lenging issue. Thus the task of constructing a canonical model structure for pectic polysaccharides has proved to be non-trivial. The structures of several individual components of pectin extracts have, however, been characterised in detail. These include three polysaccharides possessing backbones consisting of (1?4)b-D-GalA (galacturonic acid): the unsubstituted homogalacturonan (HG), the branched (1?3)-b-D-xylose-substituted xylogalacturonan (XGA) and rhamnogalacturonan II (RG-II) whose primary structure is extremely complex containing novel unique sugars and as yet not fully characterised. A proportion of the backbone galacturonic acid residues of HG and XGA are methyl esterified at C-6 and/or O-acetylated at C-2 or C-3. The degree and distribution of esterification vary according to the plant species, age and location in the plant cell wall and is implicated in controlling the extent to which gelation, a commercially important property of extracted pectins, can be induced by divalent cations such as calcium. Pectins contain rhamnose which, aside from its presence in RG-II, is found in long alternating backbone sequences consisting of the repeating disaccharide (1?2)-a-L-Rha-(1?4)-b-D-GalA in a
0008-6215/$ - see front matter Crown Copyright Ó 2010 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.carres.2009.12.019
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structure known as the rhamnogalacturonan I (RG-I) backbone.2,3 Attached to some of the rhamnose residues at C-4 are arabinogalactan (AG-I) and arabinan side chains. Arabinogalactans possess a backbone of (1?4)-b-D-galactose with a-L-arabinose attached at C-3 while the arabinans are (1?5)-a-L-arabinan polymers substituted with (1?2) and (1?3)-a-L-Ara. Collectively the RG-I backbone and the associated AG-I and arabinan are known as RG-I or ‘hairy regions’ after their putative structural arrangement. The way in which these pectic components are assembled within the plant cell wall is still not clearly understood. Recent 2D dynamic FTIR spectroscopy supports the idea that the pectin network is an independent structure interpenetrating the load-bearing cellulose-xyloglucan network.4 HG and RG-I are thought to be covalently linked as endopolygalacturonases will release RG-I regions.5 This has led to a model for ‘linear’ pectin molecules consisting of contiguous sections of HG (smooth regions) interspersed by RG-I ‘hairy regions’ (Fig. 1a). An alternative more complex branched model6 has emerged recently in which RG-I regions become a scaffold to which are attached individual HG chains (Fig. 1b). Recent structural studies of oligomers released by acid hydrolysis of pectin7 by mass spectrometry has shown that these oligomers contain covalently linked HG (and XGA) and RG-I fragments, demonstrating for the first time a covalent linkage between HG and RG-I. The nature of the oligomer, in which the RG-I was at the reducing end and the HG block at the non-reducing end of the complex, favours the contiguous model (Fig. 1a) over the more complex branched structure where the HG regions are sidechains attached to the RG-I regions (Fig. 1b). Atomic force microscopy (AFM) can produce subnanometre scale images of individual biopolymers, and has proved to be a useful tool for characterising complex, irregular and heterogeneous samples at single molecule level. AFM has been widely employed in the study of individual polymers of glycans such as cellulose,8 starch,9 mucins10,11 and a variety of bacterial polysaccharides.12,13 Pectin can be imaged by AFM and images of isolated polymers reveal the existence of branches of an unexpected morphology14,15 representing a hitherto unrecognised feature of pectin structure. This result has been supported by subsequent AFM data on pectins from marsh cinquefoil,16 tomato17 and sugar beet.17 Two pectin samples extracted from the same source (mature green tomatoes, Lycopersicon esculentum var. Rutgers) with differing levels of neutral sugars’ (arabinose and galactose) compositions both showed the same proportion of branched molecules: whilst the contour lengths and branch lengths (molecular weights) differed between
the two samples the relative proportion of the branched molecules remained similar. This observation suggested that the observed branches are not the arabinan and arabinogalactan sidechains present in the RG-I ‘hairy regions’ but are instead believed to be branched galacturonic acid backbones. As well as addressing the structures of individual molecules within pectin, the structures of gels and gel precursor fragments have been imaged by AFM.18–20 Common to these samples have been stiff, segmented, branched and kinked fibrous structures, clusters and ring-like structures, shedding some light upon the structures adopted by the individual polymers under either native or reconstituted conditions. This work demonstrates the advantage of applying techniques such as AFM to complex biopolymer systems; the morphologies of individual polymers within a population may be ascertained directly, contributing to our understanding of the structure of these complex biopolymers. It is also possible to more accurately identify the location of particular sugars within the pectin polymers by selectively removing these sugars and observing any changes that may occur in the polymer morphology.7,21 It has been shown by Thibault et al.21 that mild acid hydrolysis (0.1 M HCl, 80 °C) releases different sugar residues present in pectic polysaccharides at very different rates, with galactose and particularly arabinose linkages being most labile and the galacturonic acid being the most resistant. These authors showed that for pectins isolated from apple, beet and citrus material the principal sugars were solubilised at a decreasing rate through the series arabinose > galactose > rhamnose galacturonic acid. In addition they followed the changes in intrinsic viscosity with hydrolysis time and, since Haug et al.22 had found that the hydrolysis of alginic acid followed pseudo-first order kinetics, aimed to construct a kinetic model for the hydrolysis of pectins. They found that the data could be fitted to two pseudo-first order decays and attributed the two rates to an initial, rapid degradation of the neutral sugars arabinose, galactose and rhamnose, followed by a slower degradation of galacturonic acid. After 72 h of hydrolysis, the remaining unhydrolysed pectic material from all three sources consisted of essentially pure homogalacturonans with average degree of polymerisation (dp) of 72–100 residues, this value thus representing the minimum length of contiguous HG polymers found in pectin. In this work single molecule imaging by AFM has been combined with the sugar analysis of pectin samples extracted under alkaline conditions from mature green tomatoes and then subjected to mild acid hydrolysis. The changes in the composition of the pectic polysaccharides during mild acid hydrolysis have been correlated with the changes in the sizes and morphologies of the material observed in AFM images. The resulting changes in polymer and multipolymer complex morphology and molecular weight distributions, calculated directly from single molecule images, have then been used to examine current models of pectic polymer structure and to interpret the previous studies on the changes in intrinsic viscosity of pectin extracts on acid hydrolysis.
2. Results 2.1. Sugar analysis
Figure 1. Sketches of the two major models of pectin structure. (a) The linear contiguous model shows regions of homogalacturonan (HG) interspersed with ‘hairy regions’ of rhamnogalacturonan I (RG I) to which are attached neutral sugar sidechains such as arabinans and arabinogalactans. (b) The RG I scaffold model suggests that HG regions exist as sidechains attached to RG I alongside the neutral sugar sidechains.
The material used in this work was a 20 °C Na2CO3-soluble pectin fraction extracted from green tomato cell wall residue. The Na2CO3-extracted pectin was chosen because it contained a relatively large proportion of putative branching sugars (particularly arabinose and galactose) and because it had been subjected to de-esterification under alkaline extraction conditions. The pectin extract was subjected to mild acid hydrolysis (0.1 M HCl, 80 °C). Table 1 shows the carbohydrate composition of the starting mate-
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A. N. Round et al. / Carbohydrate Research 345 (2010) 487–497 Table 1 Sugar content of hydrolysed and unhydrolysed pectic material Hydrolysis time
% Recovered material
Rha
Unhydrolysed 0 1 4 8 24 72
86.3 76.8 75.2 69.0 39.1
0.8 1.2 1.1 1.3 0.3 0.2
Hydrolysed 1 4 8 24 72
13.7 23.2 24.8 31.0 60.9
1.6 1.3 0.9 3.5 2.5
Ara
Gal
GalA
Fuc
Xyl
Man
Glc
7.4 1.4 0.2 0.0 0.5 0.1
21.8 16.3 10.0 6.5 1.4 0.9
67.3 78.0 85.4 88.8 94.0 90.1
0.1 0.0 0.0 0.0 0.0 0.5
0.1 0.2 0.2 0.3 0.2 0.0
0.4 0.5 0.9 1.0 1.3 2.3
2.0 2.3 2.2 2.0 2.3 5.9
24.2 19.6 18.8 15.0 10.3
12.8 34.8 42.8 41.0 30.0
54.6 38.1 32.4 34.5 55.2
0.0 0.0 0.0 0.0 0.0
0.0 0.6 1.0 0.4 0.0
2.0 4.0 2.6 3.0 0.6
4.9 1.7 1.5 2.6 1.5
rial, the acid hydrolysis-resistant residue and the hydrolysate at hydrolysis times of 1, 8, 24 and 72 h. Figure 2 shows how the amounts of arabinose, galactose, rhamnose and galacturonic acid found in each sample change with hydrolysis time. It can be seen that different sugars are hydrolysed from the polysaccharide extract at differing rates, so that the composition of the extract observed by AFM varies with the hydrolysis time. Significant changes in composition were observed at different hydrolysis times for the four predominant sugars; galacturonic acid and the neutral sugars arabinose, galactose and rhamnose. The arabinose content of the residue is reduced from 7.4% in the original sample to trace amounts almost immediately: after 1 h of hydrolysis the initial content has been reduced sevenfold and after 8 h of hydrolysis it is present only at the limit of detectability. The galactose content of the residue drops at a slightly slower rate than the arabinose content. After 8 h less than 25% of the original content remains and this declines steadily during the course of the hydrolysis until after 72 h only traces remain. Loss of rhamnose from the hydrolysis-resistant polymer is not apparent at 8 h of hydrolysis but by 24 h only traces of rhamnose remain. Approximately 90% of the initial amount of galacturonic acid is retained
in the hydrolysis-resistant polymer fraction until after 72 h of hydrolysis, by which time it is reduced to 35% of its original value. The quantities of the sugars fucose, mannose, xylose and glucose remain at trace levels throughout the hydrolysis. As shown in Figure 2 the losses of sugars from the unhydrolysed residue are mirrored by the gains in the hydrolysed fraction. 2.2. AFM of hydrolysed pectin AFM images of the original Na2CO3-soluble pectin extracts were obtained, following hydrolysis in 0.1 M HCl at 80 °C for 1, 8, 24 and 72 h (Fig. 3a–d). The polymer structures observed in the images included individual strands of varying lengths and complexes (defined as structures with widths or heights greater than those measured for single strands of polymer) of varying sizes. The individual strands have diameters in the range 0.5–0.8 nm, commensurate with the expected diameters of single polysaccharide strands adopting a helical conformation when imaged by AFM,14 and lengths ranging from 20 to 400 nm. There is some variation in the widths of individual polysaccharides as shown in Figure 3a–d which probably results from differences in the tip radii for
Figure 2. Plots of the amounts of the major pectin sugars arabinose, galactose, rhamnose and galacturonic acid expressed as lg mL circles) and the unhydrolysed fraction (filled circles) sampled over the course of hydrolysis in 0.1 M HCl at 80 °C.
1
present in the hydrolysed fraction (open
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Figure 4. Plots of changes in mean backbone and branch length (filled and open circles, respectively, left-hand axis), the percentage of polymer possessing branches (open triangles) and the percentage of polymeric material present in branches (filled squares, both right-hand axis) over the course of 72 h of hydrolysis.
Figure 3. AFM images of pectin during acid hydrolysis. (a) 1 h, (b) 8 h, (c) 24 h, (d) 72 h of hydrolysis, all images 1 lm 1 lm, height 3 nm. (e–g) High resolution images of examples of complexes, all images 200 nm 200 nm, height 3 nm.
different tips used to image the molecules. The complexes range in size in all three dimensions from 1 to 10 s or even 100 s of nanometres. Figure 3e–g shows examples of these complexes, emphasising two key common features: the presence of a backbone structure in the aggregate and the emergence of one or more polymer strands resembling the individual polymer strands described above. Images of the 1-, 8- and 24-h hydrolysed samples (Fig. 3a–c) show that the polymer molecules observed possess lengths and diameters similar to those found in images obtained previously for the unhydrolysed Na2CO3-extracted pectin sample,14 but show qualitative evidence for progressively smaller complexes as the hydrolysis proceeds, suggesting that hydrolysis initially disrupts the associations that give rise to the complexes. Images of the polymers from the 72 h-hydrolysed sample (Fig. 3d) show a clear reduction in average polymer length with only a few small complexes present in the samples. The backbone length, branching and branch length distributions for individual polymers observed in each sample were recorded. Figure 4 shows how the mean backbone and branch lengths vary with hydrolysis time, together with the percentage of backbones possessing branches, and the percentage of total polymer material contained in branches for each sample over the course of the 72 h of hydrolysis. The mean backbone length does not change significantly over 24 h of hydrolysis, but after 72 h it decreases to about 50% of its 24 h value. The branch distributions show that the mean branch length remains constant over 24 h, only dropping slightly after 72 h; the branches only become degraded under conditions which cleave galacturonic acid residues, suggesting that they are homogalacturonan chains. The plots of percentage of backbones possessing branches and percentage of total polymer material
contained in branches show that the decline in the total amount of branched material is a consequence of the decline in the number of backbones possessing branches. As well as single polymers, Figure 3 shows that a significant proportion of the observed pectic materials is found to be present as aggregated complexes. In the extraction and storage of the pectic samples every effort was made to prevent aggregation: the samples were stored frozen and not freeze-dried. Hence it is believed that these complexes represent structural elements released from the cell wall material. By estimating the volumes, and hence the number-average and weight-average molecular weights of the complexes, their susceptibility to hydrolysis can be characterised in a similar way to that used for single molecules. The volume estimation protocol was originally developed by Ratcliff and Erie23 for application to protein complexes, and is described later in the methods section. The distribution of polymer lengths can also be expressed in terms of number-average and weight-average molecular weights, thereby providing direct comparisons with the results of other techniques. Figures 5 and 6 present histograms of calculated molecular weights and the constituent weight fractions for the single polymers and the complexes. Table 2 presents the number-average and weight-average molecular weights, polydispersity indices (ratios of weight-average to number-average molecular weights) and median masses alongside both hydrolysis time and the amount of neutral sugars (expressed as a percentage of all sugars) found in the unhydrolysed fraction of pectin material imaged by AFM. The polymer molecular weight distributions shown in Figure 5 and the near-constant polydispersity index of the single polysaccharide samples are consistent with a process of random single depolymerisation events resulting in two shorter daughter polymers. Molecular weights of the complexes (Fig. 6) are an order of magnitude higher than those found for the individual polymers and the polydispersity reduces significantly over the course of hydrolysis from an initial value of 3.9 to 1.2 in contrast to the behaviour exhibited by the single polymers and expected for a first order random cleavage depolymerisation process. Figure 7 corroborates this, showing that the distributions of major axis lengths (Fig. 7a) and the ratios of major to minor axes (Fig. 7b, reflecting the shapes of the complexes) are reduced both in range and mean value from their native state over the course of the hydrolysis. Despite their significantly larger masses, the lengths of the complexes’ major axes are shorter than those of individual polymers, implying that when the polymers are constituents of the com-
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Figure 5. Histograms of (a) polymer length and calculated number-average molecular weight and (b) the relative contributions of the masses of the measured polymers to the total weight-average molecular weight for individual polymers observed by AFM, as a function of hydrolysis time. The y-axis in (a) is %, in (b) it is mass fraction (kDa).
plexes they are coiled and compacted in comparison to their conformations when present in isolation. The decrease in polydispersity supports the observation that the decrease in molecular weight is caused by the breakdown of the largest complexes. The median mass of a complex in each fraction, from the unhydrolysed to the 72-h hydrolysed, remains constant at close to 380 kDa, suggesting that there is a class of complex that is unperturbed by hydrolysis. 2.3. Hydrolysis kinetics In order to more directly compare the effects of these observed morphological changes to reported studies on the rheological properties of pectin samples subjected to similar treatment, the pseudo-first order kinetics model of Thibault et al.21 was used to analyse the present data. In Figure 8, the changes in degree of polymerisation (dp, measured as number-average molecular weight MN/monomer mass m0) for single polymers (Fig. 8a) and for complexes (Fig. 8b) with hydrolysis time are shown. Figure 8a shows a broadly linear trend while Figure 8b appears to deviate significantly from linearity. Linear fits (not shown) to the two plots give R2 values of 0.91 for the plot shown in Figure 8a and of 0.80 for the plot shown in Figure 8b.
vides new insights into their structure. The task is therefore to integrate the information provided by this new view into the model of pectin structure and function constructed from the wealth of data already extant. Thibault et al.21 previously reported on changes in sugar composition and intrinsic viscosity of pectins from various sources when subjected to mild acid hydrolysis. The present studies follow a similar process but makes observations at the molecular level. Thus we can make comparisons between these datasets. Comparison of the rates at which the sugars arabinose, galactose, rhamnose and galacturonic acid are lost during hydrolysis (Fig. 2) shows that arabinose is the most labile, followed by galactose and rhamnose with galacturonic acid being the most resistant to acid hydrolysis, results which echo the findings of Thibault et al.21 This then allows a comparison of the observed changes in morphology of individual polymer structure with the changes in the sugar composition during the course of hydrolysis. By examining in detail the molecular changes that account for changes in intrinsic viscosity during hydrolysis, it is possible to propose hypotheses concerning the nature of the branching of the homogalacturonan chains, the relationship between HG and RG-I, the size of the constituent HG polymers and the overall structure of the pectin extract. 3.1. Branching in pectin
3. Discussion The ability to observe changes in the morphology of individual pectin molecules subjected to modification by acid hydrolysis pro-
Figures 3-7 show very little change during the first 24 h of hydrolysis in the backbone length and branch length distributions in individual polysaccharides. A significant reduction in backbone
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Figure 6. Histograms of (a) number-average molecular weight and (b) relative contributions of the masses of the measured complexes to the total weight-average molecular weight for complexes observed by AFM, all as a function of hydrolysis time. The y-axis in (a) is %, in (b) it is mass fraction (kDa). Note the logarithmic scale of the x-axis in (b).
Table 2 Molecular weights of polymers and complexes Hydrolysis time
% Neutral sugars in unhydrolysed fraction
Polymers 0 1 8 24 72
29.29 17.75 6.51 1.80 1.05
Complexes 0 1 8 24 72
29.29 17.75 6.51 1.80 1.05
Number-average MW (kDa) 64.3 70.1 52.3 57.9 32.6 965 690 578 500 453
length is only observed after 72 h. Thus essentially complete hydrolysis of arabinose, galactose and rhamnose has no significant effect on the structure of the individual pectin polysaccharides as visualised by AFM. Figure 9a, comparing the percentage of unhydrolysed polysaccharide containing arabinose and galactose to the percentage of individual polymers that are branched over the course of the hydrolysis, shows that the amount of branched material (9% of the total) initially accounts for less polymer material than the arabinose/galactose content (30%), and that as hydrolysis proceeds the arabinose/galactose content drops much more quickly than does the proportion of branched material. Thus the proportion of branched material is then greater than the remaining proportion of the sample consisting of arabinose and galactose. Figure 9b, comparing the changes in the rhamnose content of the
Weight-average MW (kDa) 76.5 81.2 61.7 67.5 40.5 3740 1540 1250 952 543
Median MW (kDa) 60.7 64.6 49.6 55.0 29.2 423 382 385 351 393
Polydispersity index 1.19 1.16 1.18 1.16 1.24 3.88 2.23 2.16 1.91 1.20
unhydrolysed material to changes in the ratio of branched to unbranched polymers, shows that the loss of branches does not correlate with the hydrolysis of rhamnose, as essentially all the rhamnose is lost between 8 and 24 h of hydrolysis, whereas the decrease in the level of observed branched material is most significant before 8 h of exposure to acid. These comparisons rule out the hypothesis that the branches are in fact neutral sugar sidechains. Instead the data suggest that both the linear and the branched polymers are homogalacturonans composed solely of galacturonic acid. It then becomes necessary to explain why the proportion of branched polymers decreases early during hydrolysis. There are two alternative interpretations: (i) the loss of branches occurs when the intact branches are removed at their branch point, rather
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Figure 7. Histograms of (a) major axis length and (b) ratio of major to minor axis length for complexes observed by AFM during hydrolysis. Both show a rapid loss of the longest or most elongated complexes upon hydrolysis. The y-axes in both parts are counts.
than by being degraded randomly along their length or from their non-reducing end (as the branches stay at the same length up until after 72 h of hydrolysis); (ii) new material is liberated from the degraded complexes in the form of linear, unbranched (or more sparsely branched) HG polymers and this addition effectively dilutes the proportion of branched polymers observed in the changing population. Both interpretations support the interpretation of the previous AFM studies of pectin;14,15 that the structures observed are branched HG polymers of a previously unrecognised type. The two mechanisms make different predictions about the nature of the linkage between the branches and the backbone; the first requires the existence of a branching point on the HG backbone that is markedly more labile to acid hydrolysis than the link between galacturonic acid residues in the backbone. This seems unlikely since steric considerations would suggest that the branch point would be less labile to hydrolysis. The second interpretation requires a non-uniform distribution of branched HG polymers between the isolated polymers and those captured within the complexes. There is some evidence to support this explanation since in the early stages of hydrolysis (before 1 h) the mass of the complexes decreases and the number- and weight-average molecular weights of the isolated polysaccharides increase slightly, suggesting the release from the complexes of homogalacturonan polymers with a slightly different molecular weight distribution. 3.2. Linkage between HG and RG-I The AFM data may be analysed for evidence supporting the hypothesis of a covalent linkage between HG and RG-I. In previous AFM analyses of pectin14,15 it was proposed that the neutral sugar sidechains were attached to pectin as part of the RG-I complex and were present as short branches, undetectable at the resolution
achieved by AFM. Hence it was not possible to test the linear model (Fig. 1a) by locating directly the RG-I regions. In the present work this model may be tested by observing changes (expected decreases in length) in the single polysaccharide structures when rhamnose, the key linking component of RG-I, is removed. As can be seen by comparing the mean backbone lengths in Figure 10 over the course of the hydrolysis to the changes in amounts of the neutral sugars present in the residue, removal of rhamnose is not correlated with a significant decrease in mean backbone length. The observation that the almost total removal of rhamnose after 8 h of hydrolysis does not affect the mean length of the polymer backbones, as would be expected if HG regions were interrupted by RGI regions or individual rhamnose residues along the backbone of the polysaccharide (Fig. 1a), leads to the conclusion that the observed single polymers contain either no rhamnose or only small amounts located solely at the ends of the observed polymers. Thus if any linkage between these HG structures and RG-I exists in the plant cell wall, it has to have been disrupted during the extraction of the polymers prior to imaging. The existence of the complexes, together with the fact that they reduce in size upon acid hydrolysis (Fig. 6 and Table 2) during the period when neutral sugars are lost, suggests that these may contain the RG-I fraction. A plot of complex MN against neutral sugar content (the percentage of unhydrolysed polymeric material containing arabinose and galactose), as shown in Figure 11, reveals a very good linear correlation (R2 = 0.97) between the two, and also predicts a minimum complex mass of 450 kDa (close to the 380 kDa median mass shown in Table 2). The complexes are often observed (Fig. 3e–g) to possess emergent polymer strands with dimensions commensurate in size with the isolated single polymers here identified as HG. Furthermore, as the complexes are reduced in molecular weight upon hydrolysis the mean lengths and
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Figure 8. (a) Plot following the change in degree of polymerisation of individual polymers (open circles) over the course of hydrolysis. (b) Plot following the change in degree of polymerisation of complexes (open circles) over the course of hydrolysis. The quantity D dp in the y-axes is calculated as D ln (1 m0/nMW), where m0 is the mass of a monomer and nMW is the number-average molecular weight for the unhydrolysed material at each hydrolysis time.
molecular weight distributions of the observed polymers change only slightly (Figs. 4 and 5a), suggesting that the polymeric material liberated from the complexes is similar to the isolated polymers and thus is also homogalacturonan. After 72 h of hydrolysis the remaining complexes have a MN 390 kDa. This value is close to that of a major fraction of the complexes measured in all the samples (Fig. 6) as well as to the median value (Table 2) and the intercept in the limit of no neutral sugars of the graph in Figure 11. The persistence of this fraction of complexes throughout the course of the hydrolysis suggests that it consists of a homogalacturonan resistant to prolonged acid hydrolysis. Therefore the AFM data offer evidence that the complexes are composed of homogalacturonan polymers and also stable, irreducible HG complexes, both of which may be linked to RG-I regions: both isolated HG and HG linked to RG-I are present in the Na2CO3 pectin extract. 3.3. Homogalacturonan size Thibault et al.21 measured the number-average molecular weights of the essentially pure HG remaining after 72 h of hydrolysis under similar conditions to those employed in the current study, resulting in molecular weights of the apple, beet and citrus pectins corresponding to those of the polymers of approximately 72–100 sugars. Recently Yapo et al.24 showed that pectins ex-
Figure 9. (a) Comparison between the changes in the mole fraction of unhydrolysed pectin consisting of arabinose and galactose (left-hand axis) with the percentage of measured material found in branches in the AFM images (right-hand axis). The extent and rate of loss of the neutral sugars is not reflected in the changes in the amount of branch material. (b) Comparison between the changes in the amount of rhamnose found in the unhydrolysed pectin (left-hand axis) and the percentage of individual polymers observed to possess branches (right-hand axis). Loss of rhamnose does not correlate with a reduction in branching density.
tracted from citrus peel using a range of different methods and subjected to mild acid hydrolysis also produced HG polymers with very similar molecular weights, in the range of 81–117 galacturonic acid residues (dp 81–117), and therefore hypothesised that HG polymers of this size were a common feature of pectins regardless of their location within the cell wall. Both the studies used bulk chromatographic techniques to isolate and characterise the HGs extracted. In the current study AFM allows the identification of individual polymer morphologies. In the present work the isolated polymers observed by AFM are identified as being composed of homogalacturonans, and the polymer length distributions presented in Figure 5 reflect the minimum native sizes and ranges of distribution of the HG regions in carbonate-extracted pectin before and during acid hydrolysis. Figure 5a shows that the HG polymer sizes are described by a broad distribution with a mean molecular weight corresponding to dp 320 after 24 h of acid hydrolysis, decreasing to 160 after 72 h. This increases the minimum native size estimate from the relatively narrow range of dp = 72–11721,24 to a broader range centred around dp 320, a three- or even fourfold increase on previous estimates.
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Figure 10. Comparison between the changes in the amount of rhamnose found in the unhydrolysed pectin (right-hand axis) and the mean length of polymer backbones (left-hand axis). Loss of rhamnose does not reduce the backbone lengths, suggesting that they do not contain internal RG-I regions.
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polymer lengths with hydrolysis can be compared with this interpretation. Figure 8a presents the change in the degree of polymerisation of the single polymers with hydrolysis time. A first order kinetic fit (not shown) can be made, with a single rate constant k = 3.5 10 5 h 1. The fit reflects the behaviour consistent with the evidence presented in this work, showing that the single polymers consist only of homogalacturonans, but shows some deviations at short hydrolysis times. Figure 8b is less susceptible to a linear fit, as demonstrated by the lower value of R2 for a linear fit to these data compared to that shown in Figure 8a. A qualitative assessment suggests that the initial rate of depolymerisation of the complexes is more rapid than at longer hydrolysis times, reflecting the situation described by Thibault et al.21 The evidence presented here suggests that the complexes contain both RG-I and HG so one might expect them to show two stages of hydrolysis due to breakdown of the RG-I regions and then later breakdown of HG regions. However, the kinetic models employed in that work and in this require that the breakage of a single bond produces two shorter polymers, an assumption that is valid for linear and sparsely branched polymers, but which may not hold for the degradation of complexes. Indeed we have shown in this work that the degradation of the complexes releases both HG polymers and complexes. The kinetic model of Thibault et al. does not account for this replenishment of the stock of HG polymers and thus the model is shown to be invalid in the cases of both the polymers and the complexes, even though visual inspection of Figure 8 suggests possible fits. The present studies suggest that the changes in intrinsic viscosity in the early stages of acid hydrolysis are probably due to the breakdown of complexes, and that this process cannot be modelled as a simple scission of single molecules.
3.5. Summary
Figure 11. Plot of number-average molecular weight of complexes as a function of mole fraction of arabinose and galactose in the unhydrolysed pectin. There is a good linear fit (R2 = 0.97) and the intercept of the fit in the limit of no arabinose and galactose gives a molecular weight of 450 kDa, close to the median values of the molecular weight distribution throughout the hydrolysis.
3.4. Changes in rheology (intrinsic viscosity) As well as shedding light on the structural arrangements and linkages of pectin, AFM analysis can provide a more detailed molecular picture of the changes undergone by the sample that give rise to the previously reported changes in intrinsic viscosity21 during acid hydrolysis. This can be used to assess the contributions of particular polymeric structures to the reported changes in intrinsic viscosity. Thibault et al.21 demonstrated that the decrease in intrinsic viscosity with increasing hydrolysis time showed two distinct stages, characterised by associated first order rate constants of 1–2 10 4 h 1 and 5 10 5 h 1 before and after 10 h of hydrolysis, respectively. This behaviour was attributed to two processes: Firstly a rapid hydrolysis of neutral sugar linkages leading to a rapid decline in intrinsic viscosity, followed by a slower hydrolysis of the links between galacturonic acids giving rise to a correspondingly slower decrease in intrinsic viscosity at longer hydrolysis times. As the intrinsic viscosity of a polysaccharide solution is directly related to the volume of the polysaccharide (hence the lengths or masses of the polymers) in the solution, our observation of changes in
The new view of pectin structure offered by AFM analysis of pectin extracts subjected to acid hydrolysis is depicted in Figure 12 and summarised as follows: the sodium carbonate pectin extract from unripe tomato pericarp has been shown to consist of linear and sparsely branched homogalacturonans (HG) and aggregated complexes containing rhamnogalacturonan-I (RG-I), irreducible aggregates of HG and HG polysaccharide chains. Significant amounts of individual HG polysaccharide chains either are not linked to the RG-I, or were linked through bonds that are broken during the extraction from the cell wall. On acid hydrolysis the neutral sugars and thus the RG-I polymers are degraded, leading to break up of the complexes into their constituent parts, whilst the individual HG chains, and a major fraction of the complexes, retain molecular weights similar to their initial values. After 72 h of hydrolysis only trace amounts of arabinose, galactose and rhamnose remain and the observable pectin structures consist of individual HG polysaccharides and HG aggregates. The lack of change in size of individual polymers in the event of almost total loss of arabinose, galactose and rhamnose demonstrates that these polymers consist of HG only, and therefore measurements of the size of these polymers in the freshly extracted pectin prior to hydrolysis represent a new minimum size distribution for HG polymers some three to four times larger than that previously estimated.21 4. Experimental 4.1. Polysaccharide extraction The material used in this work was a 20 °C Na2CO3-soluble fraction of pectic polysaccharide, sequentially extracted from the pericarp tissue of mature green tomatoes (Lycopersicon esculentum, var.
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to 6.5 with 1 M KOH. Chloroform was added as a bactericide. The sample was stored frozen and not freeze-dried in order to minimise any aggregation of the polysaccharides. 4.2. Acid hydrolysis 4 mL aliquots of a 1 mg mL-1 solution of Na2CO3-extracted pectin28 were acidified with 0.1 M HCl and heated to 80 °C for varying lengths of time (1, 8, 24 and 72 h). At the end of each heating phase, the sample was neutralised to pH 7 with 0.1 M NaOH and dialysed against 100 mL H2O overnight at 4 °C (MW cutoff 10 kDa). Both dialysate and residue were subjected to sugar analysis and the hydrolysis-resistant polysaccharide residue was imaged by AFM. 4.3. Sugar composition analysis Neutral sugars were released by a Seaman hydrolysis29 and reduced after neutralisation with ammonia.30 The resulting alditols were acetylated and then analysed by gas chromatography.31 The polysaccharide was subjected to carbodiimide-activated reduction of uronic acid residues,32 prior to methylation following the method of Needs and Selvendran.33 The carbodiimide reduction converts the galacturonic acid residues to deuterated galactose residues. The reduced polysaccharide was then methylated, hydrolysed, reduced and acetylated to form alditol acetates, which were analysed by GC and mass spectrometry. The original proportion of galacturonic acid was then calculated from the ratio of the deuterated to non-deuterated galactose residues. 4.4. Atomic force microscopy
Figure 12. Sketch of the distribution of pectic components present in samples of tomato pectin during progressive acid hydrolysis (a) prior to hydrolysis, (b) after 8 h of hydrolysis and (c) after 72 h of hydrolysis.
Rutgers) as described previously.25 A previous fraction, extracted using the chelating agent CDTA (15 mM, pH 4.5, 24 h) in a process designed to remove polysaccharides held in the cell wall by Ca2+mediated crosslinks,26 had already been removed. Further extraction was carried out on the cell wall residue left after CDTA extraction, using Na2CO3 at 1 °C and 20 °C, as described by Redgewell and Selvendran.27 Such conditions are believed to solubilise the pectic polysaccharides held in the wall by ester linkages. The extracted polysaccharide was stored at 20 °C as the potassium salt in aqueous solution, at a concentration of 1 mg mL 1, until required. Residual cations were removed from the acidic fraction by stirring for 20 min. over Dowex AG 50-X8 (H+) and then the potassium salt was formed by adjusting the pH of a solution of the acidic polymer
The microscope used in these experiments was constructed by East Coast Scientific (Cambridge, UK). The operation of the microscope under the conditions described below has been discussed in detail previously.14,15 For imaging, each pectin fraction was diluted to 10 lg mL 1 before the deposition of 2 lL onto freshly-cleaved sheets of mica (ca. 10 mm2). The sample was then allowed to dry under ambient conditions before insertion into the liquid cell of the microscope. 300 lL of triply distilled butanol was injected into the cell halfway through the sample approach sequence and the images were obtained in direct contact mode using cantilevers with a nominal spring constant of 0.37 Nm 1. The use of a liquid as an imaging solvent reduces the influence of capillary forces arising between the tip and the surface; butanol was chosen in particular as alcohols are used to precipitate polysaccharides from solution, thus keeping the adsorbed polysaccharide from desorbing during imaging. Topographic and error-signal mode images were collected simultaneously, and the imaging was repeated for a number of sample preparations. In excess of 100 images of areas 1– 2 lm2 were collected. For the hydrolysed samples, the concentration employed must be regarded as nominal as it is based on the concentration of the original sample and ignores the losses caused by hydrolysis; for progressively more degraded samples less visible material was expected. 4.5. Image analysis Heights of features in the images were measured using the AFM software supplied with the instrument (SPM 6.01, ECS, Cambridge, UK). For length measurements, the images were converted to TIFF files using Gwyddion (http://gwyddion.net) and analysed using ImageJ.34 Number-average and weight-average molecular weights35 were calculated assuming a monomer with molecular weight of 176 Da and a pitch per monomer of 0.435 nm.36 In order to support the use of a microscopy technique for investigating
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polymer heterogeneity at the macromolecular level, it is necessary to first demonstrate the ability to identify single polymers in a sample, and therefore to enable the rejection of complexes from subsequent analyses. AFM images allow the direct measurement of parameters in three dimensions. This ability permits the rapid identification of features with dimensions incommensurate with those expected of single polymers. It also allows the user to distinguish branches on the polymer backbone from superposition of two polymers upon each other. Measured heights of single strands vary between 0.5 and 0.7 nm between different sample preparations, due to differences in tip geometries and imaging forces, and by ±0.1 nm within an image. At a true branch point the height of the molecule will remain unchanged, whereas if two molecules cross over each other the measured height should double.15 In practice, the experimentally observed height increase at an overlap is often less than double (but always greater than 1.5) due to compression of the polymers by the AFM tip during scanning. Measuring the heights of apparent branching points ensures that superimposed polymers can be rejected from the analysis and only true branch points included. The volumes of pectin complexes were estimated using a modified version of a protocol devised by Ratcliff and Erie23 for determining protein–protein association constants from AFM images. Briefly, using the image analysis software Image SXM (http:// www.ImageSXM.org.uk), a threshold height value is set for each image defining the transition from background or single polymer to complex. The program then calculates the areas and mean heights of all complexes in the image, allowing the volumes of the complexes to be estimated. Molecular weights are then calculated from these volumes using a monomer mass of 176 Da and a volume fraction of 0.785, representing each monomer as a cylinder of 0.435 nm in length and 0.4 nm in radius.36 References 1. Willatts, W. G. T.; Knox, J. P.; Mikkelsen, J. D. Trends Food Sci. Technol. 2006, 17, 97–104. 2. McNeil, M.; Darvill, A. G.; Albersheim, P. Plant Physiol. 1980, 66, 1128–1134. 3. Zhan, D. F.; Janssen, P.; Mort, A. J. Carbohydr. Res. 1998, 308, 373–380. 4. Wilson, R. H.; Smith, A. C.; Kacurakova, M.; Saunders, P. K.; Wellner, N.; Waldron, K. W. Plant Physiol. 2000, 124, 397–405.
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5. Albersheim, P.; Darvill, A. G.; O’Neill, M. A.; Schols, H. A.; Voragen, A. G. J. In Pectins and Pectinases; Visser, J., Voragen, A. G. J., Eds.; Elsevier Science B.V.: Amsterdam, 1996; pp 47–56. 6. Vincken, J.-P.; Schols, H. A.; Oomen, R. J. F. J.; McCann, M. C.; Ulvskov, P.; Voragen, A. G. J.; Visser, R. G. F. Plant Physiol. 2003, 132, 1781–1789. 7. Coenen, G. J.; Bakx, E. J.; Verhoef, R. P.; Schols, H. A.; Voragen, A. G. J. Carbohydr. Polym. 2007, 70, 224–235. 8. Baker, A. A.; Helbert, W.; Sugiyama, J.; Miles, M. J. Biophys. J. 2000, 79, 1139– 1145. 9. Ridout, M. J.; Parker, M. L.; Hedley, C. L.; Bogracheva, T. Y.; Morris, V. J. Biomacromolecules 2004, 5, 1519–1527. 10. Round, A. N.; Berry, M.; McMaster, T. J.; Corfield, A. P.; Miles, M. J. J. Struct. Biol. 2004, 145, 246–253. 11. Round, A. N.; McMaster, T. J.; Miles, M. J.; Corfield, A. P.; Berry, M. Glycobiology 2007, 17, 578–585. 12. Kirby, A. R.; Gunning, A. P.; Morris, V. J. Carbohydr. Res. 1995, 267, 161–166. 13. Kirby, A. R.; Gunning, A. P.; Morris, V. J.; Ridout, M. J. Biophys. J. 1995, 68, 360– 363. 14. Round, A. N.; MacDougall, A. J.; Ring, S. G.; Morris, V. J. Carbohydr. Res. 1997, 303, 251–253. 15. Round, A. N.; MacDougall, A. J.; Ring, S. G.; Morris, V. J. Carbohydr. Res. 2001, 331, 337–342. 16. Ovodova, R. G.; Popov, S. V.; Bushneva, O. A.; Golovchenko, V. V.; Chizhov, A. O.; Klinov, D. V.; Ovodov, Y. S. Biochemistry-Moscow 2006, 71, 538. 17. Kirby, A. R.; MacDougall, A. J.; Morris, V. J. Carbohydr. Polym. 2008, 71, 640–647. 18. Fishman, M. L.; Cooke, P. H.; Coffin, D. R. Biomacromolecules 2004, 5, 334–341. 19. Fishman, M. L.; Cooke, P. H.; Chau, H. K.; Coffin, D. R.; Hotchkiss, A. T. Biomacromolecules 2007, 8, 573–578. 20. Morris, V. J.; Gromer, A.; Kirby, A. R. Struct. Chem. 2009, 20, 255–261. 21. Thibault, J.-F.; Renard, C. M. G. C.; Axelos, M. A. V.; Roger, P.; Crépeau, M.-J. Carbohydr. Res. 1993, 238, 271–286. 22. Haug, A.; Larsen, B.; Smidsrod, O. Acta Chem. Scand. 1966, 20, 183–190. 23. Ratcliff, G. C.; Erie, D. A. J. Am. Chem. Soc. 2001, 123, 5632–5635. 24. Yapo, B. M.; Lerouge, P.; Thibault, J.-F.; Ralet, M.-C. Carbohydr. Polym. 2007, 69, 426–435. 25. MacDougall, A. J.; Needs, P. W.; Rigby, N. M.; Ring, S. G. Carbohydr. Res. 1996, 293, 235–249. 26. Jarvis, M. C. Plant Cell Environ. 1984, 7, 153–164. 27. Redgewell, R. G.; Selvendran, R. R. Carbohydr. Res. 1986, 157, 183–199. 28. MacDougall, A. J.; Rigby, N. M.; Ring, S. G. Plant Physiol. 1997, 114, 353–362. 29. Selvendran, R. R.; March, J. F.; Ring, S. G. Anal. Biochem. 1979, 96, 282–292. 30. Englyst, H. N.; Cummings, J. H. Analyst 1984, 109, 937–942. 31. Blakeney, A. B.; Harris, P. J.; Henry, R. J.; Stone, B. A. Carbohydr. Res. 1983, 113, 291–299. 32. Needs, P. W.; Rigby, N. M.; Ring, S. G.; MacDougall, A. J. Carbohydr. Res. 2001, 333, 47–58. 33. Needs, P. W.; Selvendran, R. R. Phytochem. Anal. 1993, 4, 210–216. 34. Abramoff, M. D.; Magelhaes, P. J.; Ram, S. J. Biophoton. Int. 2004, 11, 36–42. 35. Young, R. J.; Lovell, P. A. Introduction to Polymers, 2nd ed.; Chapman & Hall, 1991. 36. Cros, S.; Garnier, C.; Axelos, M. A. V.; Imberty, A.; Perez, S. Biopolymers 1996, 39, 339–351.
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A new view of pectin structure revealed by acid hydrolysis and atomic force microscopy Andrew N. Round a,b,*, Neil M. Rigby b, Alistair J. MacDougall b, , Victor J. Morris b a b
School of Pharmacy, University of East Anglia, Norwich NR4 7TJ, UK Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, UK
a r t i c l e
i n f o
Article history: Received 3 September 2009 Received in revised form 11 December 2009 Accepted 18 December 2009 Available online 28 December 2009 Keywords: Pectin Polysaccharide Structure Kinetics Microscopy AFM
a b s t r a c t Individual pectin polymers and complexes, isolated from the pericarp of unripe tomato (Lycopersicon esculentum var. Rutgers), were subjected to a mild acid hydrolysis and visualised and characterised by atomic force microscopy (AFM). The AFM images confirm earlier studies showing that individual pectic polysaccharides often possess long branches. The AFM data have been used to construct size and molecular weight distributions for the single molecules and complexes, from which the calculated numberaverage and weight-average molecular weights can then be compared directly with the published literature data on the rheology of bulk samples. Loss of the neutral sugars arabinose, galactose and rhamnose from the pectin samples does not significantly alter either the size or the branching density of the individual polymers, but is reflected in a breakdown of the complexes. Significant loss of galacturonic acid at long hydrolysis times was found to be accompanied by changes in the size and branching of the single polymers and further breakdown of the complexes. The results suggest that rhamnose, arabinose and galactose are not the major components of the individual polymers but are, instead, confined to the complexes. The polysaccharides represent a previously unrecognised branched homogalacturonan with a minimum mean size some three times larger than that previously reported. The complexes consist of homogalacturonans (HGs) held together by rhamnogalacturonan I (RG-I) regions. Comparison of the rate of depolymerisation of the homogalacturonans and complexes with the published data on changes in the intrinsic viscosity of bulk pectin samples, subjected to similar acid hydrolysis, suggests that the different rates of depolymerisation of RG-I and HG contribute separately to the observed changes in intrinsic viscosity during acid hydrolysis. Thus data obtained using a single molecule microscopy technique provides new insights into the behaviour in the bulk. Crown Copyright Ó 2010 Published by Elsevier Ltd. All rights reserved.
1. Introduction Pectins are complex biopolymers with as yet not fully understood functions within the plant cell wall.1 Pectic polysaccharides of non-graminaceous plants are a vital structural component of the plant cell wall and pectin extracts are commercially important to the food and pharmaceutical industries; in a normal western diet around 4–5 g of pectin is consumed each day. Pectin is used in a range of processed food products as a gelling agent or as a stabiliser in acidic milk drinks. The structure of pectin varies according to the source species, original tissue type and location, developmental or metabolic stage, environmental state and conditions of extraction. This complexity and, in particular, variability of structure undoubtedly hold the key to their role(s) in the cell wall, but also make detailed analysis of pectic polysaccharides a chal* Corresponding author. Tel.: +44 (0)1603 592985; fax: +44 (0)1603 592015. E-mail address: [email protected] (A.N. Round). Present address: National Starch LLC, Bridgewater, New Jersey, NJ 08807, USA.
lenging issue. Thus the task of constructing a canonical model structure for pectic polysaccharides has proved to be non-trivial. The structures of several individual components of pectin extracts have, however, been characterised in detail. These include three polysaccharides possessing backbones consisting of (1?4)b-D-GalA (galacturonic acid): the unsubstituted homogalacturonan (HG), the branched (1?3)-b-D-xylose-substituted xylogalacturonan (XGA) and rhamnogalacturonan II (RG-II) whose primary structure is extremely complex containing novel unique sugars and as yet not fully characterised. A proportion of the backbone galacturonic acid residues of HG and XGA are methyl esterified at C-6 and/or O-acetylated at C-2 or C-3. The degree and distribution of esterification vary according to the plant species, age and location in the plant cell wall and is implicated in controlling the extent to which gelation, a commercially important property of extracted pectins, can be induced by divalent cations such as calcium. Pectins contain rhamnose which, aside from its presence in RG-II, is found in long alternating backbone sequences consisting of the repeating disaccharide (1?2)-a-L-Rha-(1?4)-b-D-GalA in a
0008-6215/$ - see front matter Crown Copyright Ó 2010 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.carres.2009.12.019
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structure known as the rhamnogalacturonan I (RG-I) backbone.2,3 Attached to some of the rhamnose residues at C-4 are arabinogalactan (AG-I) and arabinan side chains. Arabinogalactans possess a backbone of (1?4)-b-D-galactose with a-L-arabinose attached at C-3 while the arabinans are (1?5)-a-L-arabinan polymers substituted with (1?2) and (1?3)-a-L-Ara. Collectively the RG-I backbone and the associated AG-I and arabinan are known as RG-I or ‘hairy regions’ after their putative structural arrangement. The way in which these pectic components are assembled within the plant cell wall is still not clearly understood. Recent 2D dynamic FTIR spectroscopy supports the idea that the pectin network is an independent structure interpenetrating the load-bearing cellulose-xyloglucan network.4 HG and RG-I are thought to be covalently linked as endopolygalacturonases will release RG-I regions.5 This has led to a model for ‘linear’ pectin molecules consisting of contiguous sections of HG (smooth regions) interspersed by RG-I ‘hairy regions’ (Fig. 1a). An alternative more complex branched model6 has emerged recently in which RG-I regions become a scaffold to which are attached individual HG chains (Fig. 1b). Recent structural studies of oligomers released by acid hydrolysis of pectin7 by mass spectrometry has shown that these oligomers contain covalently linked HG (and XGA) and RG-I fragments, demonstrating for the first time a covalent linkage between HG and RG-I. The nature of the oligomer, in which the RG-I was at the reducing end and the HG block at the non-reducing end of the complex, favours the contiguous model (Fig. 1a) over the more complex branched structure where the HG regions are sidechains attached to the RG-I regions (Fig. 1b). Atomic force microscopy (AFM) can produce subnanometre scale images of individual biopolymers, and has proved to be a useful tool for characterising complex, irregular and heterogeneous samples at single molecule level. AFM has been widely employed in the study of individual polymers of glycans such as cellulose,8 starch,9 mucins10,11 and a variety of bacterial polysaccharides.12,13 Pectin can be imaged by AFM and images of isolated polymers reveal the existence of branches of an unexpected morphology14,15 representing a hitherto unrecognised feature of pectin structure. This result has been supported by subsequent AFM data on pectins from marsh cinquefoil,16 tomato17 and sugar beet.17 Two pectin samples extracted from the same source (mature green tomatoes, Lycopersicon esculentum var. Rutgers) with differing levels of neutral sugars’ (arabinose and galactose) compositions both showed the same proportion of branched molecules: whilst the contour lengths and branch lengths (molecular weights) differed between
the two samples the relative proportion of the branched molecules remained similar. This observation suggested that the observed branches are not the arabinan and arabinogalactan sidechains present in the RG-I ‘hairy regions’ but are instead believed to be branched galacturonic acid backbones. As well as addressing the structures of individual molecules within pectin, the structures of gels and gel precursor fragments have been imaged by AFM.18–20 Common to these samples have been stiff, segmented, branched and kinked fibrous structures, clusters and ring-like structures, shedding some light upon the structures adopted by the individual polymers under either native or reconstituted conditions. This work demonstrates the advantage of applying techniques such as AFM to complex biopolymer systems; the morphologies of individual polymers within a population may be ascertained directly, contributing to our understanding of the structure of these complex biopolymers. It is also possible to more accurately identify the location of particular sugars within the pectin polymers by selectively removing these sugars and observing any changes that may occur in the polymer morphology.7,21 It has been shown by Thibault et al.21 that mild acid hydrolysis (0.1 M HCl, 80 °C) releases different sugar residues present in pectic polysaccharides at very different rates, with galactose and particularly arabinose linkages being most labile and the galacturonic acid being the most resistant. These authors showed that for pectins isolated from apple, beet and citrus material the principal sugars were solubilised at a decreasing rate through the series arabinose > galactose > rhamnose galacturonic acid. In addition they followed the changes in intrinsic viscosity with hydrolysis time and, since Haug et al.22 had found that the hydrolysis of alginic acid followed pseudo-first order kinetics, aimed to construct a kinetic model for the hydrolysis of pectins. They found that the data could be fitted to two pseudo-first order decays and attributed the two rates to an initial, rapid degradation of the neutral sugars arabinose, galactose and rhamnose, followed by a slower degradation of galacturonic acid. After 72 h of hydrolysis, the remaining unhydrolysed pectic material from all three sources consisted of essentially pure homogalacturonans with average degree of polymerisation (dp) of 72–100 residues, this value thus representing the minimum length of contiguous HG polymers found in pectin. In this work single molecule imaging by AFM has been combined with the sugar analysis of pectin samples extracted under alkaline conditions from mature green tomatoes and then subjected to mild acid hydrolysis. The changes in the composition of the pectic polysaccharides during mild acid hydrolysis have been correlated with the changes in the sizes and morphologies of the material observed in AFM images. The resulting changes in polymer and multipolymer complex morphology and molecular weight distributions, calculated directly from single molecule images, have then been used to examine current models of pectic polymer structure and to interpret the previous studies on the changes in intrinsic viscosity of pectin extracts on acid hydrolysis.
2. Results 2.1. Sugar analysis
Figure 1. Sketches of the two major models of pectin structure. (a) The linear contiguous model shows regions of homogalacturonan (HG) interspersed with ‘hairy regions’ of rhamnogalacturonan I (RG I) to which are attached neutral sugar sidechains such as arabinans and arabinogalactans. (b) The RG I scaffold model suggests that HG regions exist as sidechains attached to RG I alongside the neutral sugar sidechains.
The material used in this work was a 20 °C Na2CO3-soluble pectin fraction extracted from green tomato cell wall residue. The Na2CO3-extracted pectin was chosen because it contained a relatively large proportion of putative branching sugars (particularly arabinose and galactose) and because it had been subjected to de-esterification under alkaline extraction conditions. The pectin extract was subjected to mild acid hydrolysis (0.1 M HCl, 80 °C). Table 1 shows the carbohydrate composition of the starting mate-
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A. N. Round et al. / Carbohydrate Research 345 (2010) 487–497 Table 1 Sugar content of hydrolysed and unhydrolysed pectic material Hydrolysis time
% Recovered material
Rha
Unhydrolysed 0 1 4 8 24 72
86.3 76.8 75.2 69.0 39.1
0.8 1.2 1.1 1.3 0.3 0.2
Hydrolysed 1 4 8 24 72
13.7 23.2 24.8 31.0 60.9
1.6 1.3 0.9 3.5 2.5
Ara
Gal
GalA
Fuc
Xyl
Man
Glc
7.4 1.4 0.2 0.0 0.5 0.1
21.8 16.3 10.0 6.5 1.4 0.9
67.3 78.0 85.4 88.8 94.0 90.1
0.1 0.0 0.0 0.0 0.0 0.5
0.1 0.2 0.2 0.3 0.2 0.0
0.4 0.5 0.9 1.0 1.3 2.3
2.0 2.3 2.2 2.0 2.3 5.9
24.2 19.6 18.8 15.0 10.3
12.8 34.8 42.8 41.0 30.0
54.6 38.1 32.4 34.5 55.2
0.0 0.0 0.0 0.0 0.0
0.0 0.6 1.0 0.4 0.0
2.0 4.0 2.6 3.0 0.6
4.9 1.7 1.5 2.6 1.5
rial, the acid hydrolysis-resistant residue and the hydrolysate at hydrolysis times of 1, 8, 24 and 72 h. Figure 2 shows how the amounts of arabinose, galactose, rhamnose and galacturonic acid found in each sample change with hydrolysis time. It can be seen that different sugars are hydrolysed from the polysaccharide extract at differing rates, so that the composition of the extract observed by AFM varies with the hydrolysis time. Significant changes in composition were observed at different hydrolysis times for the four predominant sugars; galacturonic acid and the neutral sugars arabinose, galactose and rhamnose. The arabinose content of the residue is reduced from 7.4% in the original sample to trace amounts almost immediately: after 1 h of hydrolysis the initial content has been reduced sevenfold and after 8 h of hydrolysis it is present only at the limit of detectability. The galactose content of the residue drops at a slightly slower rate than the arabinose content. After 8 h less than 25% of the original content remains and this declines steadily during the course of the hydrolysis until after 72 h only traces remain. Loss of rhamnose from the hydrolysis-resistant polymer is not apparent at 8 h of hydrolysis but by 24 h only traces of rhamnose remain. Approximately 90% of the initial amount of galacturonic acid is retained
in the hydrolysis-resistant polymer fraction until after 72 h of hydrolysis, by which time it is reduced to 35% of its original value. The quantities of the sugars fucose, mannose, xylose and glucose remain at trace levels throughout the hydrolysis. As shown in Figure 2 the losses of sugars from the unhydrolysed residue are mirrored by the gains in the hydrolysed fraction. 2.2. AFM of hydrolysed pectin AFM images of the original Na2CO3-soluble pectin extracts were obtained, following hydrolysis in 0.1 M HCl at 80 °C for 1, 8, 24 and 72 h (Fig. 3a–d). The polymer structures observed in the images included individual strands of varying lengths and complexes (defined as structures with widths or heights greater than those measured for single strands of polymer) of varying sizes. The individual strands have diameters in the range 0.5–0.8 nm, commensurate with the expected diameters of single polysaccharide strands adopting a helical conformation when imaged by AFM,14 and lengths ranging from 20 to 400 nm. There is some variation in the widths of individual polysaccharides as shown in Figure 3a–d which probably results from differences in the tip radii for
Figure 2. Plots of the amounts of the major pectin sugars arabinose, galactose, rhamnose and galacturonic acid expressed as lg mL circles) and the unhydrolysed fraction (filled circles) sampled over the course of hydrolysis in 0.1 M HCl at 80 °C.
1
present in the hydrolysed fraction (open
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Figure 4. Plots of changes in mean backbone and branch length (filled and open circles, respectively, left-hand axis), the percentage of polymer possessing branches (open triangles) and the percentage of polymeric material present in branches (filled squares, both right-hand axis) over the course of 72 h of hydrolysis.
Figure 3. AFM images of pectin during acid hydrolysis. (a) 1 h, (b) 8 h, (c) 24 h, (d) 72 h of hydrolysis, all images 1 lm 1 lm, height 3 nm. (e–g) High resolution images of examples of complexes, all images 200 nm 200 nm, height 3 nm.
different tips used to image the molecules. The complexes range in size in all three dimensions from 1 to 10 s or even 100 s of nanometres. Figure 3e–g shows examples of these complexes, emphasising two key common features: the presence of a backbone structure in the aggregate and the emergence of one or more polymer strands resembling the individual polymer strands described above. Images of the 1-, 8- and 24-h hydrolysed samples (Fig. 3a–c) show that the polymer molecules observed possess lengths and diameters similar to those found in images obtained previously for the unhydrolysed Na2CO3-extracted pectin sample,14 but show qualitative evidence for progressively smaller complexes as the hydrolysis proceeds, suggesting that hydrolysis initially disrupts the associations that give rise to the complexes. Images of the polymers from the 72 h-hydrolysed sample (Fig. 3d) show a clear reduction in average polymer length with only a few small complexes present in the samples. The backbone length, branching and branch length distributions for individual polymers observed in each sample were recorded. Figure 4 shows how the mean backbone and branch lengths vary with hydrolysis time, together with the percentage of backbones possessing branches, and the percentage of total polymer material contained in branches for each sample over the course of the 72 h of hydrolysis. The mean backbone length does not change significantly over 24 h of hydrolysis, but after 72 h it decreases to about 50% of its 24 h value. The branch distributions show that the mean branch length remains constant over 24 h, only dropping slightly after 72 h; the branches only become degraded under conditions which cleave galacturonic acid residues, suggesting that they are homogalacturonan chains. The plots of percentage of backbones possessing branches and percentage of total polymer material
contained in branches show that the decline in the total amount of branched material is a consequence of the decline in the number of backbones possessing branches. As well as single polymers, Figure 3 shows that a significant proportion of the observed pectic materials is found to be present as aggregated complexes. In the extraction and storage of the pectic samples every effort was made to prevent aggregation: the samples were stored frozen and not freeze-dried. Hence it is believed that these complexes represent structural elements released from the cell wall material. By estimating the volumes, and hence the number-average and weight-average molecular weights of the complexes, their susceptibility to hydrolysis can be characterised in a similar way to that used for single molecules. The volume estimation protocol was originally developed by Ratcliff and Erie23 for application to protein complexes, and is described later in the methods section. The distribution of polymer lengths can also be expressed in terms of number-average and weight-average molecular weights, thereby providing direct comparisons with the results of other techniques. Figures 5 and 6 present histograms of calculated molecular weights and the constituent weight fractions for the single polymers and the complexes. Table 2 presents the number-average and weight-average molecular weights, polydispersity indices (ratios of weight-average to number-average molecular weights) and median masses alongside both hydrolysis time and the amount of neutral sugars (expressed as a percentage of all sugars) found in the unhydrolysed fraction of pectin material imaged by AFM. The polymer molecular weight distributions shown in Figure 5 and the near-constant polydispersity index of the single polysaccharide samples are consistent with a process of random single depolymerisation events resulting in two shorter daughter polymers. Molecular weights of the complexes (Fig. 6) are an order of magnitude higher than those found for the individual polymers and the polydispersity reduces significantly over the course of hydrolysis from an initial value of 3.9 to 1.2 in contrast to the behaviour exhibited by the single polymers and expected for a first order random cleavage depolymerisation process. Figure 7 corroborates this, showing that the distributions of major axis lengths (Fig. 7a) and the ratios of major to minor axes (Fig. 7b, reflecting the shapes of the complexes) are reduced both in range and mean value from their native state over the course of the hydrolysis. Despite their significantly larger masses, the lengths of the complexes’ major axes are shorter than those of individual polymers, implying that when the polymers are constituents of the com-
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Figure 5. Histograms of (a) polymer length and calculated number-average molecular weight and (b) the relative contributions of the masses of the measured polymers to the total weight-average molecular weight for individual polymers observed by AFM, as a function of hydrolysis time. The y-axis in (a) is %, in (b) it is mass fraction (kDa).
plexes they are coiled and compacted in comparison to their conformations when present in isolation. The decrease in polydispersity supports the observation that the decrease in molecular weight is caused by the breakdown of the largest complexes. The median mass of a complex in each fraction, from the unhydrolysed to the 72-h hydrolysed, remains constant at close to 380 kDa, suggesting that there is a class of complex that is unperturbed by hydrolysis. 2.3. Hydrolysis kinetics In order to more directly compare the effects of these observed morphological changes to reported studies on the rheological properties of pectin samples subjected to similar treatment, the pseudo-first order kinetics model of Thibault et al.21 was used to analyse the present data. In Figure 8, the changes in degree of polymerisation (dp, measured as number-average molecular weight MN/monomer mass m0) for single polymers (Fig. 8a) and for complexes (Fig. 8b) with hydrolysis time are shown. Figure 8a shows a broadly linear trend while Figure 8b appears to deviate significantly from linearity. Linear fits (not shown) to the two plots give R2 values of 0.91 for the plot shown in Figure 8a and of 0.80 for the plot shown in Figure 8b.
vides new insights into their structure. The task is therefore to integrate the information provided by this new view into the model of pectin structure and function constructed from the wealth of data already extant. Thibault et al.21 previously reported on changes in sugar composition and intrinsic viscosity of pectins from various sources when subjected to mild acid hydrolysis. The present studies follow a similar process but makes observations at the molecular level. Thus we can make comparisons between these datasets. Comparison of the rates at which the sugars arabinose, galactose, rhamnose and galacturonic acid are lost during hydrolysis (Fig. 2) shows that arabinose is the most labile, followed by galactose and rhamnose with galacturonic acid being the most resistant to acid hydrolysis, results which echo the findings of Thibault et al.21 This then allows a comparison of the observed changes in morphology of individual polymer structure with the changes in the sugar composition during the course of hydrolysis. By examining in detail the molecular changes that account for changes in intrinsic viscosity during hydrolysis, it is possible to propose hypotheses concerning the nature of the branching of the homogalacturonan chains, the relationship between HG and RG-I, the size of the constituent HG polymers and the overall structure of the pectin extract. 3.1. Branching in pectin
3. Discussion The ability to observe changes in the morphology of individual pectin molecules subjected to modification by acid hydrolysis pro-
Figures 3-7 show very little change during the first 24 h of hydrolysis in the backbone length and branch length distributions in individual polysaccharides. A significant reduction in backbone
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Figure 6. Histograms of (a) number-average molecular weight and (b) relative contributions of the masses of the measured complexes to the total weight-average molecular weight for complexes observed by AFM, all as a function of hydrolysis time. The y-axis in (a) is %, in (b) it is mass fraction (kDa). Note the logarithmic scale of the x-axis in (b).
Table 2 Molecular weights of polymers and complexes Hydrolysis time
% Neutral sugars in unhydrolysed fraction
Polymers 0 1 8 24 72
29.29 17.75 6.51 1.80 1.05
Complexes 0 1 8 24 72
29.29 17.75 6.51 1.80 1.05
Number-average MW (kDa) 64.3 70.1 52.3 57.9 32.6 965 690 578 500 453
length is only observed after 72 h. Thus essentially complete hydrolysis of arabinose, galactose and rhamnose has no significant effect on the structure of the individual pectin polysaccharides as visualised by AFM. Figure 9a, comparing the percentage of unhydrolysed polysaccharide containing arabinose and galactose to the percentage of individual polymers that are branched over the course of the hydrolysis, shows that the amount of branched material (9% of the total) initially accounts for less polymer material than the arabinose/galactose content (30%), and that as hydrolysis proceeds the arabinose/galactose content drops much more quickly than does the proportion of branched material. Thus the proportion of branched material is then greater than the remaining proportion of the sample consisting of arabinose and galactose. Figure 9b, comparing the changes in the rhamnose content of the
Weight-average MW (kDa) 76.5 81.2 61.7 67.5 40.5 3740 1540 1250 952 543
Median MW (kDa) 60.7 64.6 49.6 55.0 29.2 423 382 385 351 393
Polydispersity index 1.19 1.16 1.18 1.16 1.24 3.88 2.23 2.16 1.91 1.20
unhydrolysed material to changes in the ratio of branched to unbranched polymers, shows that the loss of branches does not correlate with the hydrolysis of rhamnose, as essentially all the rhamnose is lost between 8 and 24 h of hydrolysis, whereas the decrease in the level of observed branched material is most significant before 8 h of exposure to acid. These comparisons rule out the hypothesis that the branches are in fact neutral sugar sidechains. Instead the data suggest that both the linear and the branched polymers are homogalacturonans composed solely of galacturonic acid. It then becomes necessary to explain why the proportion of branched polymers decreases early during hydrolysis. There are two alternative interpretations: (i) the loss of branches occurs when the intact branches are removed at their branch point, rather
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Figure 7. Histograms of (a) major axis length and (b) ratio of major to minor axis length for complexes observed by AFM during hydrolysis. Both show a rapid loss of the longest or most elongated complexes upon hydrolysis. The y-axes in both parts are counts.
than by being degraded randomly along their length or from their non-reducing end (as the branches stay at the same length up until after 72 h of hydrolysis); (ii) new material is liberated from the degraded complexes in the form of linear, unbranched (or more sparsely branched) HG polymers and this addition effectively dilutes the proportion of branched polymers observed in the changing population. Both interpretations support the interpretation of the previous AFM studies of pectin;14,15 that the structures observed are branched HG polymers of a previously unrecognised type. The two mechanisms make different predictions about the nature of the linkage between the branches and the backbone; the first requires the existence of a branching point on the HG backbone that is markedly more labile to acid hydrolysis than the link between galacturonic acid residues in the backbone. This seems unlikely since steric considerations would suggest that the branch point would be less labile to hydrolysis. The second interpretation requires a non-uniform distribution of branched HG polymers between the isolated polymers and those captured within the complexes. There is some evidence to support this explanation since in the early stages of hydrolysis (before 1 h) the mass of the complexes decreases and the number- and weight-average molecular weights of the isolated polysaccharides increase slightly, suggesting the release from the complexes of homogalacturonan polymers with a slightly different molecular weight distribution. 3.2. Linkage between HG and RG-I The AFM data may be analysed for evidence supporting the hypothesis of a covalent linkage between HG and RG-I. In previous AFM analyses of pectin14,15 it was proposed that the neutral sugar sidechains were attached to pectin as part of the RG-I complex and were present as short branches, undetectable at the resolution
achieved by AFM. Hence it was not possible to test the linear model (Fig. 1a) by locating directly the RG-I regions. In the present work this model may be tested by observing changes (expected decreases in length) in the single polysaccharide structures when rhamnose, the key linking component of RG-I, is removed. As can be seen by comparing the mean backbone lengths in Figure 10 over the course of the hydrolysis to the changes in amounts of the neutral sugars present in the residue, removal of rhamnose is not correlated with a significant decrease in mean backbone length. The observation that the almost total removal of rhamnose after 8 h of hydrolysis does not affect the mean length of the polymer backbones, as would be expected if HG regions were interrupted by RGI regions or individual rhamnose residues along the backbone of the polysaccharide (Fig. 1a), leads to the conclusion that the observed single polymers contain either no rhamnose or only small amounts located solely at the ends of the observed polymers. Thus if any linkage between these HG structures and RG-I exists in the plant cell wall, it has to have been disrupted during the extraction of the polymers prior to imaging. The existence of the complexes, together with the fact that they reduce in size upon acid hydrolysis (Fig. 6 and Table 2) during the period when neutral sugars are lost, suggests that these may contain the RG-I fraction. A plot of complex MN against neutral sugar content (the percentage of unhydrolysed polymeric material containing arabinose and galactose), as shown in Figure 11, reveals a very good linear correlation (R2 = 0.97) between the two, and also predicts a minimum complex mass of 450 kDa (close to the 380 kDa median mass shown in Table 2). The complexes are often observed (Fig. 3e–g) to possess emergent polymer strands with dimensions commensurate in size with the isolated single polymers here identified as HG. Furthermore, as the complexes are reduced in molecular weight upon hydrolysis the mean lengths and
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Figure 8. (a) Plot following the change in degree of polymerisation of individual polymers (open circles) over the course of hydrolysis. (b) Plot following the change in degree of polymerisation of complexes (open circles) over the course of hydrolysis. The quantity D dp in the y-axes is calculated as D ln (1 m0/nMW), where m0 is the mass of a monomer and nMW is the number-average molecular weight for the unhydrolysed material at each hydrolysis time.
molecular weight distributions of the observed polymers change only slightly (Figs. 4 and 5a), suggesting that the polymeric material liberated from the complexes is similar to the isolated polymers and thus is also homogalacturonan. After 72 h of hydrolysis the remaining complexes have a MN 390 kDa. This value is close to that of a major fraction of the complexes measured in all the samples (Fig. 6) as well as to the median value (Table 2) and the intercept in the limit of no neutral sugars of the graph in Figure 11. The persistence of this fraction of complexes throughout the course of the hydrolysis suggests that it consists of a homogalacturonan resistant to prolonged acid hydrolysis. Therefore the AFM data offer evidence that the complexes are composed of homogalacturonan polymers and also stable, irreducible HG complexes, both of which may be linked to RG-I regions: both isolated HG and HG linked to RG-I are present in the Na2CO3 pectin extract. 3.3. Homogalacturonan size Thibault et al.21 measured the number-average molecular weights of the essentially pure HG remaining after 72 h of hydrolysis under similar conditions to those employed in the current study, resulting in molecular weights of the apple, beet and citrus pectins corresponding to those of the polymers of approximately 72–100 sugars. Recently Yapo et al.24 showed that pectins ex-
Figure 9. (a) Comparison between the changes in the mole fraction of unhydrolysed pectin consisting of arabinose and galactose (left-hand axis) with the percentage of measured material found in branches in the AFM images (right-hand axis). The extent and rate of loss of the neutral sugars is not reflected in the changes in the amount of branch material. (b) Comparison between the changes in the amount of rhamnose found in the unhydrolysed pectin (left-hand axis) and the percentage of individual polymers observed to possess branches (right-hand axis). Loss of rhamnose does not correlate with a reduction in branching density.
tracted from citrus peel using a range of different methods and subjected to mild acid hydrolysis also produced HG polymers with very similar molecular weights, in the range of 81–117 galacturonic acid residues (dp 81–117), and therefore hypothesised that HG polymers of this size were a common feature of pectins regardless of their location within the cell wall. Both the studies used bulk chromatographic techniques to isolate and characterise the HGs extracted. In the current study AFM allows the identification of individual polymer morphologies. In the present work the isolated polymers observed by AFM are identified as being composed of homogalacturonans, and the polymer length distributions presented in Figure 5 reflect the minimum native sizes and ranges of distribution of the HG regions in carbonate-extracted pectin before and during acid hydrolysis. Figure 5a shows that the HG polymer sizes are described by a broad distribution with a mean molecular weight corresponding to dp 320 after 24 h of acid hydrolysis, decreasing to 160 after 72 h. This increases the minimum native size estimate from the relatively narrow range of dp = 72–11721,24 to a broader range centred around dp 320, a three- or even fourfold increase on previous estimates.
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Figure 10. Comparison between the changes in the amount of rhamnose found in the unhydrolysed pectin (right-hand axis) and the mean length of polymer backbones (left-hand axis). Loss of rhamnose does not reduce the backbone lengths, suggesting that they do not contain internal RG-I regions.
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polymer lengths with hydrolysis can be compared with this interpretation. Figure 8a presents the change in the degree of polymerisation of the single polymers with hydrolysis time. A first order kinetic fit (not shown) can be made, with a single rate constant k = 3.5 10 5 h 1. The fit reflects the behaviour consistent with the evidence presented in this work, showing that the single polymers consist only of homogalacturonans, but shows some deviations at short hydrolysis times. Figure 8b is less susceptible to a linear fit, as demonstrated by the lower value of R2 for a linear fit to these data compared to that shown in Figure 8a. A qualitative assessment suggests that the initial rate of depolymerisation of the complexes is more rapid than at longer hydrolysis times, reflecting the situation described by Thibault et al.21 The evidence presented here suggests that the complexes contain both RG-I and HG so one might expect them to show two stages of hydrolysis due to breakdown of the RG-I regions and then later breakdown of HG regions. However, the kinetic models employed in that work and in this require that the breakage of a single bond produces two shorter polymers, an assumption that is valid for linear and sparsely branched polymers, but which may not hold for the degradation of complexes. Indeed we have shown in this work that the degradation of the complexes releases both HG polymers and complexes. The kinetic model of Thibault et al. does not account for this replenishment of the stock of HG polymers and thus the model is shown to be invalid in the cases of both the polymers and the complexes, even though visual inspection of Figure 8 suggests possible fits. The present studies suggest that the changes in intrinsic viscosity in the early stages of acid hydrolysis are probably due to the breakdown of complexes, and that this process cannot be modelled as a simple scission of single molecules.
3.5. Summary
Figure 11. Plot of number-average molecular weight of complexes as a function of mole fraction of arabinose and galactose in the unhydrolysed pectin. There is a good linear fit (R2 = 0.97) and the intercept of the fit in the limit of no arabinose and galactose gives a molecular weight of 450 kDa, close to the median values of the molecular weight distribution throughout the hydrolysis.
3.4. Changes in rheology (intrinsic viscosity) As well as shedding light on the structural arrangements and linkages of pectin, AFM analysis can provide a more detailed molecular picture of the changes undergone by the sample that give rise to the previously reported changes in intrinsic viscosity21 during acid hydrolysis. This can be used to assess the contributions of particular polymeric structures to the reported changes in intrinsic viscosity. Thibault et al.21 demonstrated that the decrease in intrinsic viscosity with increasing hydrolysis time showed two distinct stages, characterised by associated first order rate constants of 1–2 10 4 h 1 and 5 10 5 h 1 before and after 10 h of hydrolysis, respectively. This behaviour was attributed to two processes: Firstly a rapid hydrolysis of neutral sugar linkages leading to a rapid decline in intrinsic viscosity, followed by a slower hydrolysis of the links between galacturonic acids giving rise to a correspondingly slower decrease in intrinsic viscosity at longer hydrolysis times. As the intrinsic viscosity of a polysaccharide solution is directly related to the volume of the polysaccharide (hence the lengths or masses of the polymers) in the solution, our observation of changes in
The new view of pectin structure offered by AFM analysis of pectin extracts subjected to acid hydrolysis is depicted in Figure 12 and summarised as follows: the sodium carbonate pectin extract from unripe tomato pericarp has been shown to consist of linear and sparsely branched homogalacturonans (HG) and aggregated complexes containing rhamnogalacturonan-I (RG-I), irreducible aggregates of HG and HG polysaccharide chains. Significant amounts of individual HG polysaccharide chains either are not linked to the RG-I, or were linked through bonds that are broken during the extraction from the cell wall. On acid hydrolysis the neutral sugars and thus the RG-I polymers are degraded, leading to break up of the complexes into their constituent parts, whilst the individual HG chains, and a major fraction of the complexes, retain molecular weights similar to their initial values. After 72 h of hydrolysis only trace amounts of arabinose, galactose and rhamnose remain and the observable pectin structures consist of individual HG polysaccharides and HG aggregates. The lack of change in size of individual polymers in the event of almost total loss of arabinose, galactose and rhamnose demonstrates that these polymers consist of HG only, and therefore measurements of the size of these polymers in the freshly extracted pectin prior to hydrolysis represent a new minimum size distribution for HG polymers some three to four times larger than that previously estimated.21 4. Experimental 4.1. Polysaccharide extraction The material used in this work was a 20 °C Na2CO3-soluble fraction of pectic polysaccharide, sequentially extracted from the pericarp tissue of mature green tomatoes (Lycopersicon esculentum, var.
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to 6.5 with 1 M KOH. Chloroform was added as a bactericide. The sample was stored frozen and not freeze-dried in order to minimise any aggregation of the polysaccharides. 4.2. Acid hydrolysis 4 mL aliquots of a 1 mg mL-1 solution of Na2CO3-extracted pectin28 were acidified with 0.1 M HCl and heated to 80 °C for varying lengths of time (1, 8, 24 and 72 h). At the end of each heating phase, the sample was neutralised to pH 7 with 0.1 M NaOH and dialysed against 100 mL H2O overnight at 4 °C (MW cutoff 10 kDa). Both dialysate and residue were subjected to sugar analysis and the hydrolysis-resistant polysaccharide residue was imaged by AFM. 4.3. Sugar composition analysis Neutral sugars were released by a Seaman hydrolysis29 and reduced after neutralisation with ammonia.30 The resulting alditols were acetylated and then analysed by gas chromatography.31 The polysaccharide was subjected to carbodiimide-activated reduction of uronic acid residues,32 prior to methylation following the method of Needs and Selvendran.33 The carbodiimide reduction converts the galacturonic acid residues to deuterated galactose residues. The reduced polysaccharide was then methylated, hydrolysed, reduced and acetylated to form alditol acetates, which were analysed by GC and mass spectrometry. The original proportion of galacturonic acid was then calculated from the ratio of the deuterated to non-deuterated galactose residues. 4.4. Atomic force microscopy
Figure 12. Sketch of the distribution of pectic components present in samples of tomato pectin during progressive acid hydrolysis (a) prior to hydrolysis, (b) after 8 h of hydrolysis and (c) after 72 h of hydrolysis.
Rutgers) as described previously.25 A previous fraction, extracted using the chelating agent CDTA (15 mM, pH 4.5, 24 h) in a process designed to remove polysaccharides held in the cell wall by Ca2+mediated crosslinks,26 had already been removed. Further extraction was carried out on the cell wall residue left after CDTA extraction, using Na2CO3 at 1 °C and 20 °C, as described by Redgewell and Selvendran.27 Such conditions are believed to solubilise the pectic polysaccharides held in the wall by ester linkages. The extracted polysaccharide was stored at 20 °C as the potassium salt in aqueous solution, at a concentration of 1 mg mL 1, until required. Residual cations were removed from the acidic fraction by stirring for 20 min. over Dowex AG 50-X8 (H+) and then the potassium salt was formed by adjusting the pH of a solution of the acidic polymer
The microscope used in these experiments was constructed by East Coast Scientific (Cambridge, UK). The operation of the microscope under the conditions described below has been discussed in detail previously.14,15 For imaging, each pectin fraction was diluted to 10 lg mL 1 before the deposition of 2 lL onto freshly-cleaved sheets of mica (ca. 10 mm2). The sample was then allowed to dry under ambient conditions before insertion into the liquid cell of the microscope. 300 lL of triply distilled butanol was injected into the cell halfway through the sample approach sequence and the images were obtained in direct contact mode using cantilevers with a nominal spring constant of 0.37 Nm 1. The use of a liquid as an imaging solvent reduces the influence of capillary forces arising between the tip and the surface; butanol was chosen in particular as alcohols are used to precipitate polysaccharides from solution, thus keeping the adsorbed polysaccharide from desorbing during imaging. Topographic and error-signal mode images were collected simultaneously, and the imaging was repeated for a number of sample preparations. In excess of 100 images of areas 1– 2 lm2 were collected. For the hydrolysed samples, the concentration employed must be regarded as nominal as it is based on the concentration of the original sample and ignores the losses caused by hydrolysis; for progressively more degraded samples less visible material was expected. 4.5. Image analysis Heights of features in the images were measured using the AFM software supplied with the instrument (SPM 6.01, ECS, Cambridge, UK). For length measurements, the images were converted to TIFF files using Gwyddion (http://gwyddion.net) and analysed using ImageJ.34 Number-average and weight-average molecular weights35 were calculated assuming a monomer with molecular weight of 176 Da and a pitch per monomer of 0.435 nm.36 In order to support the use of a microscopy technique for investigating
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polymer heterogeneity at the macromolecular level, it is necessary to first demonstrate the ability to identify single polymers in a sample, and therefore to enable the rejection of complexes from subsequent analyses. AFM images allow the direct measurement of parameters in three dimensions. This ability permits the rapid identification of features with dimensions incommensurate with those expected of single polymers. It also allows the user to distinguish branches on the polymer backbone from superposition of two polymers upon each other. Measured heights of single strands vary between 0.5 and 0.7 nm between different sample preparations, due to differences in tip geometries and imaging forces, and by ±0.1 nm within an image. At a true branch point the height of the molecule will remain unchanged, whereas if two molecules cross over each other the measured height should double.15 In practice, the experimentally observed height increase at an overlap is often less than double (but always greater than 1.5) due to compression of the polymers by the AFM tip during scanning. Measuring the heights of apparent branching points ensures that superimposed polymers can be rejected from the analysis and only true branch points included. The volumes of pectin complexes were estimated using a modified version of a protocol devised by Ratcliff and Erie23 for determining protein–protein association constants from AFM images. Briefly, using the image analysis software Image SXM (http:// www.ImageSXM.org.uk), a threshold height value is set for each image defining the transition from background or single polymer to complex. The program then calculates the areas and mean heights of all complexes in the image, allowing the volumes of the complexes to be estimated. Molecular weights are then calculated from these volumes using a monomer mass of 176 Da and a volume fraction of 0.785, representing each monomer as a cylinder of 0.435 nm in length and 0.4 nm in radius.36 References 1. Willatts, W. G. T.; Knox, J. P.; Mikkelsen, J. D. Trends Food Sci. Technol. 2006, 17, 97–104. 2. McNeil, M.; Darvill, A. G.; Albersheim, P. Plant Physiol. 1980, 66, 1128–1134. 3. Zhan, D. F.; Janssen, P.; Mort, A. J. Carbohydr. Res. 1998, 308, 373–380. 4. Wilson, R. H.; Smith, A. C.; Kacurakova, M.; Saunders, P. K.; Wellner, N.; Waldron, K. W. Plant Physiol. 2000, 124, 397–405.
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