J. Mol. Biol. (1982) 155, 517-531 Conformations II. Influence I). A. (Received of Pectins of Residue Sequence on Chain Association Calcium Pectate Gels E. POWELL, Pnilever and Interactions R. MORRIS. M. J. GINEY AXI) I). A. in REES Research. Colworth Laboratory, Sharnbrook Bedford MK44 ILQ, England 19 May 1981, and in revised form 16 November 1981) In the accompanying paper (Morris et al., 1982) we present evidence of Ca’+induced association of poly-o-galacturonate sequences from pectin into dimers of 2, chain symmetry, with co-operative (“egg-box”) binding of Ca2+ on specific sites along the interior faces of each chain. We now investigate the role in calcium pectate gel networks of other structural features, in particular methyl esterification and 1,2-linked L-rhamnosyl residues in the polymer backbone. Acid hydrolysis of citrus, apple and sunflower pectins gave polygalacturonate blocks with a relatively narrow molecular weight distribution, and average chainlength of -25 residues in each case. Since the known relative stabilities of glycosidic linkages would lead to chain cleavage predominantly at L-rhamnose, this result indicates that the length of polygalacturonate sequences between rhamnose interruptions is approximately constant within and between the pectins studied. Calcium pectate gel strength is reduced dramatically by the incorporation of these chain segments when they are de-esterified, but not when they are esterified. This interference with the development of a network structure that resists applied stress, provides further support for our model of junction zone formation from sequences of contiguous deesterified residues, with Cazf -mediated chain dimers providing the primary associations that can offer resistance to deformation. Samples with different levels and patterns of esterification were prepared by enzymic (blockwise) and chemical (random) de-esterification of almost fully methyl esterified pectin. In the former series, the extent of Ca2+ binding (as monitored by circular dichroism) increased almost linearly with the fraction of free carboxyl groups, whereas the latter showed a nonlinear relationship of a form consistent with the requirement of this binding for blocks of contiguous non-esterified residues and, in the presence of excess univalent cations, binding was negligible when more -40% of the carboxyl groups were esterified. Statistical calculations of than sequence length distribution at different degrees of random de-esterification show the best fit with experimental data when binding is assumed to require sequences with seven or more consecutive free carboxyl groups along the participating face of the chain. For 2, chain symmetry, this corresponds to a sequence length of 14 residues, in excellent agreement with previous independent studies of Ca’+ binding to oligogalacturonates. In the absence of competing univalent counterions, circular dichroism changes o are similar in form but so large in magnitude that site-binding of Ca*+ mustriow Ca f+ beyond the half-stoichiometry at which it is arrested in their presence. 517 (M,Z2-2836/82/080517-15 $02.00/O C 1982 Academic Press Inc. (London) Ltd 518 I). A PO\VEl,l. ET AL. binding monitored by circular dichroism. and gel strength (yield stress) measured mechanically. both show a similar dependence on the I)attern as well as the Ipvel of esterification. as expected for network formation by co-operative binding of (*a*+ within interchain junction zones. To fit this binding data cluant,itatively, it is necessary to postulate a two-stage process. (1) Initial dimerization, probably corresponding to the “stjrong associations” indicated by evidence from rompetitive inhibition (see above). for which a critical minimum sequence of seven residues is again required but rsterified residues can now be accommodated within individual sites provided that they are paired with a free carboxylate on the complementary chain. (2) Subsequent (‘a’+induced aggregation of these preformed dimrrs, which ran occur irrespective of the pattern of esterification on the external faces; the evidence from mechanical measurements shows that these contribute little to gel strength a.t high stress. 1. Introduction Studies of the gelation behariour of pol~vsacc+arides i~r vitro. after extraction and purification, have proved a valuable and experimentally convenient route to molecular understanding of their structural role ir, viw (see. for example. Rees, 1977: Rees & Welsh, 1977: Morris et nl., 1977: Stockton et al., 1980). A unifying principle that has now emerged from investigation of many differerlt gelling polysaccharide systems is that interchain association occurs by the formation of extended, conformationally regular “junction zones”. We have shown (Morris et nl., 1978) that the primary event in the calciurninduced gelation of alginate, t.he principal polysaccharide component. of marine brown algae (Phaeophyeeae). is dimerization of poly-L-guluronate chain sequences. These pack as buckled, 2-fold ribbons (Mackie. 1971) with polar cavities or pockets. which provide an array of regularly spaced, specific sites for cation chelation (Grant et al.. 1973). In view of the obvious analogies, we have adopted the term “egg-box” binding to denote cation-mediated interchain associations of this type. Pectin is an important constituent of the cell wall of higher plants, and is based, like alginate. on a linear 1,4-linked polyuronate backbone, in this case of n-u-galacturonate. The poly-u-galacturonate sequences of pectin and the poly-L-guluronate sequences from alginate are almost exact mirror images, and we have presented evidence in the accompanying paper (Morris et al.. 1982) that Ohis structural parallel extends to their association with calcium, including closely analogous 2, egg-box geometry. Although the formation of stable int.ermolecular junctions is a critical requirement for gelation, some limitation of t,he extent of interchain association is also necessary to give a hydrated network rat)her than an insoluble precipitate. In the case of alginate, for example. dimeric poly-L-guluronate junction zones are terminated by the occurrence in the primary sequence of n-mannuronate, either in homopolymeric blocks, or in mixed sequences with L-guluronate (Smidsrod. 1974: Morris et al., 1978). Our principal objective in the present work has been to investigate covalent features that have a similar solubilizing function in pertic polysaccharides. Pectin? unlike alginate, is based on a single uranic acid residue. and this ma) occur either as the free salt, or as the methyl ester. We have therefore explored the effect, on gelation behaviour of the extent. and pattern of esterification. Other (‘.dLCIl’M I’ECTXTE GELS 519 st,ruct,ttrat feat)ures with clear implications for the restriction of interchain association are side-chains of neutral sugar residues, and also the occurrettce of 1.2. linked L-rhamnosyl residues as covalent. insertions in the polymer backbone 1965: Gould rt al.. 1965; AAspitiall et ~1.. (Xspinatt, 1970: Zitko 6 Bishop, 1967.1968). It is not known whether these rhamttosyl insertions are 1 or p-linked. Ilut computer model-building calrulat,iotts (Rees $ Wight. 1971 ) show that’ both art incompatible wit,h regular conformations of Ftol?r-D-galacturottate, and wouttl therefore act as junction delimiting “kinks”. analogous to those identified in t-he agar and carrageenatt series (Rees, 1969.1972). It. should be emphasized thatt 1 :‘tlinked rhamnosyl residues do not. of course, have a universal kinking futtction. Thus. in one of the Klebsieh polpsaccharides (KS serotype), where such residues has form part of t,he regular repeating sequence. an ordered chain conformation lieen characterized in the solid state (Atkins rt crl., 1979), while an analogous 1.2. linked I)-mattttosgl residue that forms part of t,he petttasaccharide repeating structure of xattthatt is essential for adopt,iott of the ordered conformation, b\ allowing side-chains to fold compactly alottg the polymer backbone (Moorhouse rl (11.. 1977). Ln the specific case of pectin, however. incorporation of such residues within ordered conformations of the fl(‘lSgala~turotlate chain is stericatt) impossible. not because of any general or ittherent propert,y of 1 .&linked rhamttost (cf. Xt.kitts et txl.. 1979). but. because t.tte linkage is di#~~nt from that in the rest of the Ilolymer backbone. In t,his work we have investigated the distribution of rhatnnose kinks along the polymer backbone. and have compared our results kvitlt the known critical sequence length (Kahn, 1975) for the formation of stable calcium E”lt~galacturottat,e interchain junctions. ,4 prelimittar~ account, of part of this work has been published elsewhere (Gidley st al., 1979). 2. Experimental The followitq samples were used: I. 11 from Bulmers Ltd.. Hereford. U.K.. wvert unstandardized citrus pect.itts and had degrees of esterification (fraction of urotta.tr residues that are methyl esterified) of 36’?,, and 7 l?lh, respectively ; 111 (“Brown ribbon“). at1 apple ptvtitt from Obipectitt, contained 72 oh ester: IV, V were sunflower pectins (kindly donated sutttlower by Dr B. de Vries), and had ester contents of 440/; and 53%, respectively. Thra pectins were supplied pure as freeze-dried powders. Commercial pectin samples w(arp precipitated from aqueous solution with ethanol, washed with acidified aqueous ethanol (5(?;, (v/v) HCI in 60% (v/v) aqueous ethanol) and dialysed extensively against deionized water before being freeze-dried. E&r and urottate values were determined either by ittfra-red spectroscopy (Bociek & Welti, 1975) or by saponification and titration. (II) Prepardion qf pectin hlockn Pectin (1 p) was dissolved in deionized wat,er (80 ml) and diluted with hydrochloric acid to give a final concentration of about I(+<, (u/v) polysaccharide in 05 M-acid. The solution was heated on a boiling waterbath for 3 h : a precipitate normally appeared after about 1 h. The hydrolvsate was cooled, centrifuged and. after neutralization. soluble and insoluble fractions bvere dtalysed separately and freeze-dried. Blocks were fractionated on a column of Sephadex (XX) elut,ed with deionized water, and were typically obtained in art overall yield of -(iP,, 520 Il. A. POWELL (see Table 1). For the competitive inhibition were used; after de-esterification these chromatography columns was monitored phenol/sulphuric acid assay (Dubois ft al., (c) Preparation ET AI, experiment,, both samples behaved by optical rotation 1956). and soluble and insoluble identically. Elution measurement,s or blocks from by the USC of pectinestrrasc Pectinesterase was extracted from oranges, essentially according to the method of McDonnell et al. (1945), and purified by chromatography on DEAE-cellulose (Datunashvili et al., 1976). Enzyme activity was detected in column fractions by the following assay: 1 ml samples were added to a solution (1 ml : 1 sb, w/v) of pectin of high ester content (samples II or III). The solution was then adjusted to pH 7.0, and allowed to stand at ambient temperature (-25°C). Release of free carboxyl groups was detected by a drop in pH, typically within 5 to 10 min, and monitored by titration with NaOH (01 M). The purified enzyme was found to be low in pectin chain splitting activity, as monitored by measurement of the intrinsic viscosity of the de-esterified samples (capillary viscometer: @2 M-NapI). Samples having different levels of ester subst,itution were prepared enzymicallg from pectin with a degree of esterification of 920’ ,0 (prepared from pectin 11 as below). Pectin (1.5 g) in 0.1 M-NaCl was adjusted to pH 7.0 and mixed with 100 ml of a solution containing pectinesterase (40 mg) in @I M-NaCl at pH 7.0, and digested at 25°C. The release of free carboxyl groups was monit,ored by continuous Ctration with @l iv-NaOH. At, suitable times, samples were removed and quenched by precipitation of the enzyme with an equal volume of 10% (w/v) trichloroacetic acid. The samples were filtered. dialysed and freeze-dried. Although de-est,erification occurred more rapidly at higher pH, the reaction was carried out at neutrality to avoid concurrent base-catalpsed saponification. (d) Alkalinr dr-astrr<Jicatiorr of p&in This method was used to prepare ester-free pectin blocks for t,he competitive inhibition experiment, and polymeric samples having different levels of ester arranged in random distribution. The conditions were chosen to minimize chain cleavage by fl-elimination. An @59; (w/v) aqueous solution of pectin was maintained at 1°C’ on an ice/salt bath, and adjusted to pH 12.0 with 2 M-NaOH. The pH was maintained by the periodic addition of alkali and. after suitable times, samples were removed. quenched in acid, dialvsed and freeze-dried. Intrinsic viscosity measurements showed that chain cleavage was mmimal. Gel strengths were measured on cylindrical plugs of gel (height 12 mm, diam. 12.5 mm) using an Instron Universal Materials Tester. The method used for preparation of gels by slog release of (:a’+ from an insoluble salt. was typically bv addition of a solut,ion of citric acid (0.446 g in 10 ml water) with rapid stirring to a solution (40 ml) containing pectin (0.5 g). sodium citrate (@44 g) and calcium orthophosphate (0.143 g). The mixture was poured immediately into moulds, and aged for at least 12 h before measurement. An alternative method of gelation was by mixing a solution of pectin (300 mg in 20 m1) at. 8O”f’ with a solution of calcium chloride (132 mg of the dihydrate in 10 ml) at the same temperature : gels were again formed in cylindrical moulds and aged for at least 12 h. The latter method was used only at pH 3 and below: at more alkaline pH values, a gel formed as soon as the solutions were mixed. When gels were made for rompetitive inhibition studies, pectin blocks were added with the pectin. (‘ircular dichroism (Morris et al., 1982). measurements The neutral sugar of solution content, and gels were made as described of pectin samples was determined before by gas- (‘ALCIVM PECT.4TE GELS *521 liquid chromatography analysis of alditol acetates, using an internal standard (Bjiirndal of nl., 1970), after hydrolysis with sulphuric acid (0.25 M, lOO”(‘, 16 h). The chain length of pectin blocks was estimated from the molar ratio of reducing end groups, determined by t.he method of Park & ,Johnson (1949), to the total uronate content determined by infra-red spectroscopy (Bociek Sr Welti. 1975). All samples were de-esterified before end group analysis, to avoid possible p-elimination under the alkaline conditions used in the assay procedure. Highly esterified (tvpically 84-950;,) pectins were prepared by treatment of pectin II (12.7 g) \vith 1 M-methanoiir sulphuric acid for 70 h at 2°C”. Samples were recovered by washing ethanol and ether. soquent,ially u ith NF;, ( r / L-) a q ueous ethanol until neutral, absolute 3. Acid Hydrolysis of Pectin When pectins (samples I to V) were exposed to strong mineral acid (@25 MH,SO,) at lOO”(’ they were initially soluble, but material was precipitated after one to t)wo hours. After three hours. the mixtures were cooled, and separated by c+ent,t=ifugation into soluble and insoluble fractions, both of which had high uronate cont)ent. low levels of neutral sugars, and were almost fully de-esterified. the residual degree of esterification being in the range 0 to 5% in all cases. The soluble fractions from all samples studied showed essentially identical profiles on column chromatography (Sephadex G-SO), and eluted as a single, sharp included peak (Fig. l), indicating a narrow range of molecular weight. A sharp peak in the same position was observed as t,he major hydrolysis product in the insoluble fractions. but some material of higher molecular weight was also present (Fig. 1). \I:r at)tribute the separation into soluble and insoluble fractions to precipitation of this higher molecular weight material, with some incorporation of shorter chains within t’he aggregate structure, while t)he major hydrolysis product is of sufficiently low molrcular weight to remain soluble even when fully de-esterified. Results fol Froctlon number PI<:. 1. Sephadex G-50 fractionation of (0) neutralized acid insoluble and (a) soluble blocks from pectin I. The column (400 mm x 26 mm) was held at 20°C. and eluted with distilled water at a flow-rate of 9 ml/h. Column loading was -0.4 g of dialysed and freeze-dried hydrolysate. Fractions of 4-5 ml each were collected, and monitored by optical rotation (578 nm: 10 cm pathlength). V, z 80 ml: V, z 330 ml: elution volume at band centre ~175 ml. Similar elution profiles were obtained for the soluble and ,nsolnble fractions of all pectins studied. 522 D. A. POWELL TABLE Percentage Pectin yields ET B.C. 1 of polygalacturolbate blocks on hydrolysis Soluble Insoluble blocks of pectins blocks both yield and degree of polymerization were consistent from run to run, and a similar fragmentation pattern was observed for all the samples studied (Table 1). Glycosidic linkages of uranic acid residues are known to be largely resistant to acid hydrolysis at pH values sufficiently low to induce protonation of the carboxy groups (Lindberg et al., 1975). Polyuronate chain sequences of pectin would therefore be expected to remain essentially intact under such conditions. Neutral sugar linkages and, in particular. those of furanose sugars or 6-deoxy hexoses, however, are considerably more labile. The observed hydrolytic scission of pectin would therefore suggest removal of neutral sugar side-chains, and cleavage at the Lrhamnosyl insertions that are known to occur in the polymer backbone (Gould et al., 1965; Aspinall et al., 1967,196s). Although acidic hydrolysis cannot. of course, be absolutely specific, and the presence of some residual material of higher molecular weight indicates incomplete scission of rhamnosyl linkages. nonetheless the narrow range of chainlengths in the major hydrolysis product, and its isolation in comparatively high yield (typically -700/;, of the insoluble fraction, and -85’j/, of the total hydrolysis product), would suggest that the uninterrupted polygalacturonate chain sequences have a relatively narrow distribution of lengths in the native polymer. Estimation of the chainlength of this major product, by assay for reducing endgroups (Park & Johnson, 1949) gave an average degree of polymerization of -35 Since t,he latter using galacturonic acid as standard. and -25 using L-rhamnose. seems the more appropriate standard, we conclude that the regular, uninterrupted sequence length in all the pectin samples studied is approximately 25 residues. 4. Competitive Inhibition of Pectin Gelation Competitive inhibition is widely used to study the specificity of biological recognition events, for example, enzyme-substrate or lectirr-hapten binding. This approach has been extended to characterization of chain-chain interactions in regular chain gelling polysaccharides (Morris et al., 1980). using structurally segments to occupy binding sites on intact polymer chains, and thus inhibit the formation of a crosslinked network. Thus, for example, when poly-r-guluronate blocks, isolated by partial hydrolysis of alginate, are added to gelling concentrations of the parent molecule, calcium-induced gelation is inhibited, while poly-n-mannuronate and heteropolymeric mixed blocks have little effect, in C’ALC’TUM Concentratm PIG. 2. Effect of added pectate blocks (slow release of Ca* + ) : (0) (yelation from heated solution). blocks 84% PECTATE of added 523 GELS blocks (X, w/v) on calcium pectate (1 O/o (w/v) gel strength). esterified blocks (slow release of C’a* + ) : (A) (0) De-esterified de-esterified blocks agreement with previous evidence that cation binding and inter-chain association are largely restricted to polyguluronate chain sequences. We have therefore used this approach to probe the primary structural requirements for junction zone formation in calcium pectate gels. I’oly-r-galacturonate blocks, prepared by controlled hydrolysis of pectin, were de-esterified by mild saponification, and purified by gel filtration. Two alternative gelation procedures were used. The first of these involved addition of an insoluble calcium salt to the polysaccharide solution, and subsequent controlled release of calcium ions by addition of acid. In the second method, calcium chloride was added to the polysaccharide solution at 8O”C, and gelation occurred on cooling to ambient temperature. Gels prepared by the first technique were consistently weaker, but, this is probably due to inclusion of air, or mechanical disruption of the incipient network. during the final mixing step when acid is added. In both experiments, the presence of polygalacturonate blocks had an inhibitory effect on gel strength. measured as the stress required to rupture the sample (yield stress). Similar large reductions were observed in the maximum deformation that could be accommodated by the network before failure (breaking strain). As shown in Pigure 2, the reduction in gel strength increases progressively with increasing concentration of polygalacturonate chain segments. until network structure is almost completely lost at a block concentration approximately half that of the intact polymer. The presence of esterified blocks (84% of uronate residues methyl esterified), by contrast, was found (Fig. 2) to have no significant effect on gel strength. 5. Effect of Degree and Pattern of Esterification Studies of Ca2+ activity degrees of polymerization in the presence of polygalacturonate chains of different (Kohn. 1975: Kohn & Luknar, 1977) indicate a critical 524 I>. A. PO\VEI,I, E7’ .4/,. chainlength requirement of about 11 residues for co-operative binding. For the longer chains studied here. this need for a critical sequence lengt,h should be seen for junction zone formation in calcium-induced gelation. What is not obvious. however. is whether esterified residues can be accommodated within the junctions. or whether they will act as jllrlction-termirlatillg structural features. in the same way as kinking residues. To explore this further, UY have examined the gelation behaviour of pectin samples n-it,h both different levels and different patSterns of methyl esterification. and under different conditions of ionic euvironmr~tlt Since thr distribut,ion of esterified residues in nat,ural pectins will bc det.ermined both by partial saponification during the rxtraction procedure (whicah xvi11 bcb essent,ially random) and by the pre-existing distribution in tlirv (which is probAt\. syst,ematir). we first. prepared material t.ha.t was almost c~omplctely methyl rsterified ( -9Os,,) and then partially de-&erified using both chemical and enzymic, methods. Alkaline hydrolysis is known t,o remove ester groups in a random manner. and will thus gilye pectins wit.h an a.pproxima.tely stat.istical distribution of free carboxyl groups. 13~ contrast, pectinesterase initiates its a&ion adjacent to a free rarboxyl group, and proceeds along the chain in a stepwise fashion. producing blocks of free carboxyls (Kohn rf cl/.. 196%: Itexova-Bcnkova & Markovic,. 1978). Starting from highly esterified material, therefore. the enzq’tnic method should yield pectins in which blocks of est.erified residues alt.rrnat~e Lvith blocks of fret galact,uronate. The characterist.ic circular dichroism chmgcs ((irant et (1.1.. 1973: Morris et c/l.. 1973 : Bryce rt ul.. 1974) that. accompany calcium pectate gelation arv seen onIF for co-operative binding of Ca* + . and not, for simple electrostatic int,eractions with polygalaeturonate chain sequences that are too short for co-operatirity (Kohn KT Sticzay, 1977; Bytrick? rt al... 1979). We have therefore used this technique to quantify the extent of junction zone formation in our various pectin samples. a,nd structural requirements for stable interchain hence to identify the primary association. Ln t,he ac~companying paper (Morris vt (I/.. 19X2) lye argued that calciurnmediated association of unesterified polygalacturonatt chains in thp presence of swamping levels of competing univalent ca.tions is limited to t,hc formation of 2, chain dimers. Under these ionic conditions. the extent of (‘a’+ binding. as monitored by circular dichroism change (Fig. 3). decreases dramat.icalty with increasing content of esterified residues randomly distributed aloug the polymer chain. The form oft he observed dependence of (‘a* + binding capacit)y WI degree of random esterification suggest,s that the format’ion of stable interchain junctiotl zones requires participation of uninterrupted sequences of free carboxyl groups. To test and quantify this hypothesis. we have compared our experimental results with st,atistical calculations of sequencr length distribution. If the fraction of esterified galacturonat~e residues isJ’. then the rjrobability of u norl-esterified residues occurring consecutively along one face of the polymer chaiu is (t -f)“. As shown in Figure 4, a value of IL = 7 closely matches the obse~~~~etl dependence. This corresponds to a minimum vhaiulength of II residues in a chaiu of 2, symmetry, in excellent agreement with t,he t,hreshold value of 12 to 16 residues determined for co-operat,ive binding of (‘a’ + by galacturonate oligomers in free (‘ALCIITM PE(‘TATE Degree of esterhotlon GELS 525 (%I FIN:. 3. Effect of degree and pattern of esterification on the calcium binding capacity of pectin. as monitored by circular dichroism. (0) Enzymically de-esterified pectin: free availability of Ca’+ : (0) alkaline de-esterified pectin; free availability of Ca’+ : (A) alkaline de-esterifed pectin ; Ca* + binding in competition with 0.5 M-Xa+, In the latter 2 cases. the continuous lines show the best fit obtained from comparison of the primary sequence requirements for co-operative binding of Ca*+ postulated in the text. and statistical calculations of the distribution of esterified residues. cd.. circular dichroism. solut,ion (Kohn. 1975). consistent with dimerization of 2, helices as previously proposed (Morris et al.. 1982). These results indicate that co-operative binding of (‘a*+ in competition with excess univalent counterion requires a minimum critical sequence length of seven unesterified galacturonate residues, and is terminated b> the occurrence in the primary structure of an esterified residue on either of the participating chain faces. l’nder more normal gelation conditions, where Cazf is present as the sole OI principal counterion, the extent of Ca*+ binding to unesterified polygalacturonate, as monit’ored by circular dichroism (Fig. 3). is approximately twice as great as in the competitive situation discussed above. We attribute this to extensive dimerr dimer aggregation, with carboxyl groups along both sides of each 2, chain participating in cation chelation, rather than only those on the interior surfaces of isolated dimers. thus doubling overall Pa’+ binding capacity. In the absence of excess amounts of competing urlivalent cat.ions, gels prepared from pectin samples of low ester content are highly turbid. This is reflected in the increased scatter of our circular dichroism data for these systems (Fig. 3). and provides further evidence of extensive aggregation. I’nder these conditions of ionic environment. Ca2+ binding capacity shows (Fig. 3) a sigmoidal dependence on the degree of random esterification, with little reduction in the magnitude of circular dichroism change until approximately onethird of t,he galacturonate residues are esterified. From this we conclude (1) that there is some limited tolerance for esterified residues within the ordered, interchain ,junctions. a.nd (2) that ester groups incorporated in this way experience the same 526 1). A. POWELL ET A/, 200 0 Fractm of non-esterifled residues ( f) Fit:. 4. Variation in cat,ion binding to t)ol~galacturorlatr with degree of random esterification. bound calcium that is resistant to displacement by swamping levels of monovalent counterion Sa’), as monitored by circular dichroism (r,d.) change (a) between solution and gel (see Morris 1982) is compared with the statistical probability of occurlp~~cc of 6 (- - -), 7()or8(---,-) consecutive nowesterified residues along one chain face. for a wage of methyl polygalacturonatcs. The (@5 Met crl.. electronic perturbations from the r~lf’orcw~ proximit,.; of site-bound calcium ions. to make the same molar contribut,ion as free carboxyl groups to the overall magnitude of circular dichroism change. To explore further the primary seyuence requirements for the formation of stable interchain junctions in the absence of excess univalent couuterions, we have again attempted to match t’he observed dependence of the magnitude of circular dichroism change on the degree of random esterification by calculation of the probability of occurrence of specific dist,ributions of esteritied and unesterified residues. This is clearly a more complex analysis than the previous calculations of the distribution of consecutive unesterified residues. and a number of seemingly attract,ive models were tested, but failed to reproduce the observed dependence. .A very good fit (Fig. 3) was obtained. howrvrr. on the basis of the following simple postulates. (1) (2) A minimum critical sequence length is again required for co-operativity. In the initial dimerization process. calcium ions may occupy egg-box nest)s in which one or both of the participating carboxyl groups are unesterified. but ,jurlctiorl-zone formation is t#erminated when rsterified residues on both chains coincide. (‘AL(‘II:M (3) PE(‘TATE Subsequent extensive dimer-dimer residue sequence along the outer 527 GELS aggregation faces. occurs irrespective of the If we again denote the fraction of esterified galacturonate asf, and consider the growth of a pre-existing dimer, then the probability that the next residue on both participating chains is esterified (thus terminating the junction) is f2. Hence the probability of any other combination, to extend the junction, is (1 -f2). Since the initial residue pairing is a function of the relative positioning of the chains, rather than their primary sequence, the probability of occurrence of a dimeric junction zone of IL residues along each chain face and with no disallowed coincidence of ester, groups is (1 -f’)“-‘. Subsequent association of all such dimers into large aggregates will then increase the fraction of residues participating in (:a’+ binding t.0 2(1 -f’)“V with the proviso that the maximum value can never, of course. exceed unity. (This proviso takes account of the fact that at low levels of esterification the outer faces of dimeric junctions may also satisfy the sequence criteria for inner faces.) In terms of this model. the best fit (Fig. 3) to the experimental data is obtained for TL = 7, as previously found for (:a’+ binding in competition with excess univalent cations. Although success in matching observed results in terms of a particular model does not, of course, establish unequivocally the validity of the model, we regard the excellent agreement of the critical sequence length calculated for both sets of ionic conditions studied with previous independent investigations (Kohn. 1975) as additional compelling evidence in support of t,he proposed interpretation. For pectin samples prepared by enzymic (blockwise) de-esteritication. the magnitude of circular dichroism change 011 addition of Cazf (Fig. 3) was approximately proportional to the fraction of free carboxyl groups, confirming the need for consecutive un-esterified galacturonate residues. We further conclude that the polygalacturonate chain sequences form egg-box junctions in the same way as isolated polygalacturonate blocks. and the fully esterified sequences take no part in Degree and FIG. 5. Gel strength as a function (0) alkaline de-ester&d pectin. of degree of esterification of esterification b&o) for (e) enzymically de-esterified pectin. 528 D. A. POWELL ET -4L. the binding mechanism. By this circular dichroism criterion, the extent of junction zone formation in block (enzymically) de-esterified pectin samples is greater t,han in randomly de-esterified materials when the degree of esterification exceeds - 154,, To test this conclusion. we have compared the mechanical strength (yield stress) of calcium pectate gels prepared from materials of both types. The threshold degree of est,erilication for the formation of gels with a measurable yield stress (Fig. 5) is for “block de-esterified” pect,ins. and - 50% for randomly -SD/b de-esterified samples. For the former series, yield stress increases approximately linearly wit’11 decreasing ester level below this threshold value. while the latter show much greater sensitivity to reduct,ion of ester content, below 5OS,. Xs shown in Figure 5. yield stress measurements indicate greater interchain association for the block deesterified samples at degrees of esterification above -15?;,, in good agreement wit’h the circular dichroism evidence. 6. General Discussion In the accompanying paper (Morris et al.. 1982) we present evidence that’ the primary mechanism of calcium-induced association of poly-o-galacturonate chain sequences is by dimerization, closely analogous to that previously demonstrated (Morris et al.. 1978) for poly-L-guluronate sequences of alginat,e. In this respect. pectin and alginate may therefore be regarded as courlterparts in certain land and sea plants, respectively. III the present work, we have shown that this analogy extends to competitive inhibition of network formation by chain segments identical in chemical structure to the principal binding regions of the parent molecules (polyu-galacturonate and poly-r-guluronate. respectively). In both cases. the major fur&on of other chain sequences present in t,he molecule (totally or predominantly esterified regions in pectin. and homopolymeric or heteropolymeric regions involving o-mannuronate in alginate) appears to be in solubilization and hydration of the network, and these sequences show no such competitive inhibition of interchain association. It has been shown (see Cook & Stoddart, 1973) that at an early stage in the biosynthet,ic pathway pectin exists in an essentially fully esterified form. and the pectin t,hat is most readily extracted from mature tissue also typically shows high degrees of esterification ( -709/,). The presence in plant tissue (Gould et 01.. 1965: Rees & Wight. 1969: Cook & Stoddart. 1973) of pectic material that can be or by chemical degradation. extracted only by the use of Ca2+ sequestrants, however, indicates that. some chains have undergone substantial de-esterification. which is believed to occur at a later stage of biosynthesis. by the action of pectinesterase. and would give rise to a blockwise arrangement of free carboxy groups. Our present results explain why this product would indeed form tight. (‘a2+-mediated associations in the plant tissue. Biosynthesis of structural polysaccharides as soluble precursors that, are subsequently converted to the structure-forming species by enzymic modification at the polymer level has also been demonstrated for the agar (Rees, 1961), carrageenan (Lawson & Rees, 1970) and alginate (Larsen & Haug, 1971; Haug & Larsen, 1971) families, and may represent a common mechanism for biological regulation of tissue structure. (‘AL(‘JI’M PEC’TATE GELS 529 In both alginate and pectin. selective binding of calcium in competition with much higher concentrations of univalent cations is terminated by the occurrence in the primary sequence of residues typical of the sotubilizing stretches (methyl gatacturonate. or D-matttturonate. respectively). While such conditions are relevant to the external ionic environment in oi~o for alginate, the same is not true for pectin. Therefore. it is perhaps significant t,hat, white mattnuronate is incompatible with interchain association even in the absence of competing counterions. it appears from our present results that some degree of methyl esterification can then be accommodated within calcium pectate juttctiott zones. This may be as high as about, a third of the participating residues. provided t,hat they are distribut,rd randomly along the chain. rather than grouped in homopolymeric blocks. The covalent feature in the primary structure of pectin that exerts the same absolute delimiting effect’ on junctiott-zone length in pectin as I)-mattnuronatt does in alpinat,e is the insertion in the polymer backbone of 1 .&linked L-rhamttosyt residues. which have been shown by computer model-building (Rees 8 Wight. 197 1 ) to be incompatible with inclusion in conformationally regular F”)t?lgata~turortate junctions. In this work. we have shown that the spacing of these is such that the tbngth of kinking residues along the polymer backbone uttinterrupt.ed sequences of n-galacturottate (free salt or ester) does not vary widely bvithin or between t,he samples we have examined. and is around 25 residues. (‘CC operat.ive binding of (“a’+, irrespective of ionic erivirotitnetit. appears to require the ittvolvemettt of at least seven residues along each of t’he participating chain faces. bvhirh for Z-fold symmetry corresponds to a critical sequence length of about 11 residues. Thus. any appreciable reduct~ion in the spacing of rhamttose insertions would he likely to comprotnise the stability of ittt,erchain association. white wider spacing would decrease the tot,al number of junctions. The natural dist.ributiott of rhamttose kinks itt pectin thus represent’s an efficient (possible optimum) balanc~c brtwern the number of ittterchaitt associations and their stability. Such periodic strrtc~turat irregularities in the polymer backbone might also offer art explanation of t)hr pronounced structural periodieity visualized directly in electron micrographs of pect,itr stained \\ith rut,hrttium red (Hanke B Northcote. 1975). For l)oth algitta.te (Morris rt 01.. 1!)7X) and pectin (Morris rf (rl.. 198:!), the concentration of competing mortovalrttt countcriotts required to rest,rict ittterchaitr associa,tion to egg-box dimers is 0.5 %I. Under these conditions. t,he critical primar? strucatural requirement for the formation of’ stable calcium pectate junction zones apl)ears to be seven consecutive free earboxyt groups along the interior faces of each of the participating chains. The same crit,ical srquenre length appears to persist in the presence of (ia’+ as the sole or principal counterion. but in t,his case t~strrified residues can be accommodated within t’he dimer structure. provided that thr corresponding residue on the other chain is not also esterified. Our results suggest that under these ionic conditions extensive ditner-dimer aggregation ttta? o(~~tr. irrespecbtive of the residue sequence along the exterior faces. This lack of specificity. and ease of disruption by competing cations, indicates that the associa,tiotts btxtween dimers are far weaker and more t,ettuous than those bet,ween participating chains within dimers. Further support for this conclusion comes from the abilit’y of short chain segments to sigttificant,ly reduce the strength of gel 530 D. A. POWELL ET AL. networks, even under ionic conditions where there is evidence of substantial aggregation, since only interactions of the latter type will be inhibited (Morris et al., 1980). We may therefore regard the calcium pectate gel structure as a strong, cohesive network cross-linked by dimeric egg-box junctions between unesterified chain faces which, under appropriate ionic conditions, may be reinforced by weaker associations of partially esterified chain sequences to form additional, less stable dimers, and by dimer-dimer aggregation. Under the experimental conditions used in this work, fully or predominantly esterified chain sequences appear to have no structural role other than as interconnecting, solubilizing stretches between junctions, although the gelation of pectins of high ester content at low pH values and reduced water activity is well-known. and utilized commercially. In summary, the pectin chains in our samples contain blocks of n-galacturonate approximately 25 residues in length, joined together by sequences that contain 1.2. linked L-rhamnosyl insertions. Since the geometry of these anomalous residues is incompatible with the regular 2-fold conformation required for interchain association in egg-box junctions, they act as an invariant restriction on chain packing, to prevent total insolubility. The extent and pattern of methyl esterification exerts a more subtle, regulatory function, which may be of importance (Rees. 1969) for differentiation, growth and consolidation of tissue structure. We thank providing the Mr E. J. Murray samples for experimental of sunflower assistance, and Dr B. de Vries for kindly pectin. REFERENCES Aspinall, G. 0. (1970). Polysaccharides, Pergamon Press, Oxford. Aspinall, G. O., Cottrell, 1. W., Egan, S. V., Morrison, I. M. & Whyte, J. N. C. (1967). J. Chem. Sot. ser. C, 1071-1080. Aspinall, G. O., Craig, J. W. T. & Whyte, J. L. (1968). Carbohdr. Res. 7, 442-452. Atkins, E. D. T., Isaac, D. H. & Elloway, H. F. (1979). In Microbial Polysuccharides and Polysacchurases (Berkeley, R. C. W., Gooday. G. W. & Ellwood, D. C., eds), pp. 161~ 189, Academic Press, London. Bjiirndal, H., Hellerqvist, C. G., Lindberg, B. & Svenson! S. (1970). Angew. Chem. Znt. Ed. Engl. 9, 610-619. Bociek, S. M. & Welti, D. (1975). Carbohydr. Res. 42, 217-226. Bryce, T. .4., McKinnon, A. A., Morris, E. R., Rees, D. A. & Thorn, D. (1974). Faraday Lhcuss. Chem. Sot. 57, 221-229. Bystricki, S., Kohn, R. & Sticzay, T. (1979). Collect. Czechoslovac. Chem. Commun. 44, 167173. Cook. G. M. W. & Stoddart, R. W. (1973). Surface Carbohydrates of the Eukaryotic Cell, Academic Press, London. Datunashvili, E. K’., Tyurina, S. S. & Kardash, N. LJ. (1976). Appl. Biochem. Microbial. 12, 36-39. Dubois, M., Gillies, K. A., Hamilton, J. K., Rebers, I’. A. & Smith, F. (1956). ilnal. Chem. 28. 350-356. Gidley, M. J., Morris, E. R., Murray, E. *J., Powell. D. A. B Rees, D. A. (1979). J. Chem. Sot. Chem Commun. 990-992. Gould, S. E. B., Rees. D. A., Richardson, N. G. & Steele. 1. W. (1965). LVature (London), 208, 876-878. C*AL(‘IUM (:rant, G. T., Morris. E. R., Rees, PECTATE D. A., Smith, GELS P. J. C. & Thorn, 531 D. (1973). FEBS Letters. 32. 195198. Hanke. D. E. & Northcote, D. H. (1975). Biopolymers, 14, 1-17. Haug. A. B Larsen. B. (1971). Biochim. Biophys. Acta. 192, 557-559. Kohn. R. (1975). Pure Appt. Chem. 42. 371-397. Kohn. R. & Luknar, 0. (1977). Collect. Czechostovac. Chem. Commun. 42, 731-744. Kohn, R. 8r Sticzay, T. (1977). Collect. Czechoslovac. Chem. Commun. 42, 2372-2378. Kohn. R., Furda, I. XL Kopec, Z. (1968). Collect. Czechoslovac. Chem. Commun. 33, 264-269. Larsen. B. & Haug, A. (197 1). Carbohydr. Res. 20, 225-232. Lawson. C’. J. & Rees, D. A. (1970). ,Vat~re (London). 227. 392393. Lindbcrp. B.. Lonngren, *J. & \,‘vesson, S. (1975). =Idvan. Carhohydr. Chem. 31, lSeF-235. JlcDonnell. L. It.. Jansen, E. F. & Lineweaver, H. (1945). rlrch. Bioch,em. 6, 3899401. hfackie. W’. (1971). Biochem. J. 125, 89Y. Moorhouse, It.. Walkinshaw. X. D. & Arnott, S. (1977). In E:ctracellular j!Iicrobiu./ Polysaccharidrs, llCS Symp. ser (Sandford, P. A. & Laskin, A., eds), vol. 45, pp. 90-102. AC‘S, W’ashington. Morris. E. R., Rees, D. A. 8r Thorn, D. (1973). J. Chem. Sot. Chem. Gommun., 245-246. Morris. E. R., Rers, D. A.. Thorn, D. & Welsh, E. ,J. (1977). J. Supramol. Struct. 6, 259-274. Morris, E. R.. Rers, D. A., Thorn, D. & Boyd. J. (1978). Carbohydr. Res. 66, 145-154. Morris. E. R.. Revs. D. A., Robinson: G. & Young, G. i\. (1980). J. Mol. Biol. 138, 363-374. JIorris, E. R., Powell, D. A.. Gidley, M. J. & Rees, D. A. (1982). J. Mol. Biol. 153, 507-516. Park. .J. T. 8~ Johnson, M. J. (1949). J. Biol. Chem. 181, 149-151. Rres. D. A (1961). Biochem. J. 81, 347-352. Rem I). A., (1969). Advan,. Carhohydr. Chem. Biochem. 24, 267-332. Reea. I). A. (1972). Biochem. J. 126, 2577273. kes. D. A. (1977). Polysaccharide ShapcCs, Chapman & Hall, London. Rres. D. A. 8: Welsh. E. J. (1977). =Inqcrc, Chem. Int. Ed. Engl. 16, 214-224. Rcrs. 1). -4. & Wight. A. W. (1971). J. Chem. Sot. ser. B, 1366-1371. Rexova-Benkova. L. & Markovic, 0. (1976). Advan. Carhohydr. Chrm. 33, 323-385. Smidsrod. 0. (1974). Faraday Discuss. Chem. Sot. 57, 263-286. St,ocbkt,on, B., Evans, L. V., Morris. E. R.: Powell, D. A. bt Rees, D. A. (1960). Botanica Marina Zitko. 23 563-567. c. T. (1965). V. & ‘Bishop, Canad. J. Chem. Edited by 43, 32063214. V Luzznti
0
You can add this document to your study collection(s)
Sign in Available only to authorized usersYou can add this document to your saved list
Sign in Available only to authorized users(For complaints, use another form )