863Bsochem. J. (1969)114, 863Printed in Great Britain

Solution Properties of Polygalacturonic Acid

By R. W. STODDART,* I. P. C. SPIRES AND K. F. TIPTONDepartment of Biochemi8try, University of Cambridge

(Received 2 May 1969)

1. The specimen of polygalacturonic acid used in these studies was shown tocontain very little neutral sugar, methyl ester groups or ash, and only residues ofgalacturonic acid. Its electrophoretic homogeneity was examined in pyridine-aceticacid buffer at pH6-5 and in borate buffer at pH9-2. The distribution of effectiveparticle weights was shown to be fairly narrow. 2. The pH-titration curve of thepolymer gave a pK value of 3-7. 3. The interaction of the polymer with RutheniumRed was studied and titration curves were obtained for the spectral shifts associatedwith the formation of a complex. 4. Optical-rotatory-dispersion studies showedthat the Drude constant, A,, was dependent on pH. 5. Polygalacturonic acid wasshown to display non-Newtonian properties in solution and to have an anomalouslyhigh relative specific viscosity atlow concentrations. 6. Studiesweremade ofthepH-dependence of the sedimentation coefficient of the polymer. 7. These results arediscussed in terms of the structure of the molecule and their relevance to theproperties of pectic substances.

Pectinic acids were long considered to be poly-galacturonic acids in which a proportion of thecarboxyl groups was esterified, and theories of theirrole in cell development stressed the importance oftheir free-carboxyl-group content and its effect inbinding Ca2+. In particular, Ca2+ was thought toform bridges between adjacent chains of low estercontent. Studies by Schweiger (1962, 1963, 1964,1966) have shown that the binding of Ca2+ bypolygalacturonic acids is intramolecular rather thanintermolecular in solution (unlike binding by alginicacid) and involves the hydroxyl groups of the poly-mer in addition to its carboxyl groups. Moreover,since the demonstration by Aspinall & Fanshawe(1961) of the covalent linkage of neutral sugars topectinic acids, numerousworkers have found neutralsugars in pectinic acids, mostly in the form ofneutral'blocks' (Barrett & Northcote, 1965; Aspinall,Begbie, Hamilton & Whyte, 1967; Aspinall, CottrelEgan, Morrison & Whyte, 1967; Aspinall, Hunt &Morrison, 1967). The studies by Stoddart, Barrett& Northcote (1967) and Stoddart & Northcote(1967) have further shown that pectinic acids canexist both with and without neutral blocks and thatthese blocks are metabolically related to the pecticarabinogalactans. Consequently the simple modelof the function of pectins is no longer tenable andany new model that is developed must take intoaccount more subtle and complex features of the

* Present address: Medical Research Council MolecularPharmacology Research Unit, Old Press Site, Mill Lane,Cambridge.

physical chemistry of the molecule. The presentstudy of polygalacturonic acid is an approach to aninvestigation of the properties of these molecules,by way of a simpler molecule related to them.

MATERIALS AND METHODSPolygalacturonic acid (derived from citrus pectin:

mol. wt. range 2 x 104-6 x 104) was purchased from Koch-LightLaboratories Ltd., Colnbrook, Bucks. All other chemi-cals were obtained from British Drug Houses Ltd., Poole,Dorset, or Hopkin and Williams Ltd., Chadwell Heath,Essex, and were of the highest purity available. Themolecular weight of Ruthenium Red was calculated fromthe formula as given in the Merck Index (Stecher, 1968). Thetechniques for a determination of moisture and ash, thepreparation of pectin pectyl hydrolase (EC 3.1.1.11) andenzymic de-esterification have been described by Barrett &Northcote (1965). (Methyl a-D-galactopyranosid)uronicacidwasprepared bythe method ofMorell & Link (1933).

Chromatographic 8eparation of neutral augar8. This wasperformed on Whatman no. 1 paper with ethyl acetate-pyridine-water (8:2:1, by vol.) (Jermyn & Isherwood,1949). The sugars were detected by the aniline hydrogenphthalate method (Wilson, 1959).

Zone electrophore8i8 of the polygalacturonic acid. Electro-phoresis was carried out on Whatman GF 81 glass-fibrepaper. Separations were performed either in pyridine-acetic acid buffer, pH6-5 [10% (v/v) pyridine, 0.3% (v/v)acetic acid, + EDTA, final concn. 10mM] or in sodiumtetraborate buffer, pH9.2 (0.05M). For separations atpH6-5 a potential gradient of 44v/cm. was used for 25min.at 200 in a tank similar to that described by Michl (1959),with toluene containing 4% (v/v) of pyridine as coolant.For separations at pH9-2, a potential gradient of 40v/cm.

R. W. STODDART, I. P. C. SPIRES AND K. F. TIPTONwas applied for 30 min. at 200 in a tank ofthe type describedby Durrum (1950), with white spirit as the coolant. Bothtypes of tank were cooled by circulation of water througha cooling coil immersed in the coolant; both had platinumelectrodes.

Electrophoretic zones were located by means of thesulphonated 1-naphthol reagent described by Barrett &Northcote (1965).

Detection of amino acid8 on electrophoretogram8 and in8olution. Thepresence ofaminoacids on electrophoretogramswas investigated by spraying with indanetrione hydrate andtheir presence in solutions of polygalacturonic acid wasassayed by the method ofStein & Moore (1948).

Hydroly8is ofpoly8accharide8. Hydrolyses were performedwith sulphuric acid and the hydrolysates were neutralizedwith N-methyl-NN-di-n-octylamine (Stoddart et al. 1967).Neutralized hydrolysates were analysed by paper chromato-graphy.

E8terification and reduction of polygalacturonic acid.Polygalacturonic acid was esterified with ethylene oxide asdescribed by Barrett & Northcote (1965) and Stoddart &Northcote (1967). The partially esterified product wasreduced with KBH4 (Stoddart & Northcote, 1967).pH mea8urement8. These were made with a Radiometer

type 22 pH-meter. Measurements were made at 18°. Titra-tion curves were determined with a Radiometer TTT IcpH-stat at 18°. The concentration of polygalacturonicacid used was either 0.5% or 1-0% (w/v). NaOH (1-0M)was used as the titrant and was standardized immediatelybefore use. Titration curves were also determined for 1-0%(w/v) polygalacturonic acid in the presence of 1 mm-Ruthenium Red, 0-25m-KCI or 01M-lsodium phosphate.Blank titrations were performed in all cases. For allexperiments adjustment of the pH of the polygalacturonicacid solution was made with 0 5m-HCI or 0 5m-NaOH addedvery slowly in the form of drops to a vigorously stirredsolution ofthe polysaccharide.

Vi8co8ity mea8urement8. These determinations were madeat 18° in 0-1 m-potassium phosphate buffer, pH 6-5, over theconcentration range 0-025-2% (w/v). A horizontal capillaryviscometer (Tsuda, 1928; Ostwald, 1933) was used with anadjustable pressure-head. The capillary diameter was0-042 cm. and the length 24-795 cm. The viscometer waslevelled before use and each measurement was made at aconstant pressure.

Analytical ultracentrifugation. The sedimentation of a1% (w/v) polygalacturonic acid solution in 0 2M-potassiumphosphate buffer was observed in a Beckman-Spinco modelE ultracentrifuge. Schlieren photographs were taken at16min. intervals after attainment of top speed(59780rev./min.). The temperature was maintained at 200.Runs were performed at various pH values and concentra-tions. The pH of the solution was adjusted to the requiredvalue immediately before the start ofthe run. Sedimentationcoefficients were calculated by the graphical method ofMarkham (1960).

Partial 8pecific volume. The partial specific volume of thesodium salt of the polygalacturonic acid was measured in apycnometer of 0-74ml. capacity at 200.

Spectrophotometric analy8e8. Spectra of polygalacturonicacid and Ruthenium Red and the adduct of the two weredetermined in a Unicam SP. 800 recording spectrophoto-meter at 250. Comparison of the spectra ofthe polymer anddye before and after mixing was facilitated by the use of

cm.

Fig. 1. Design of the 'split' spectrophotometer cell. Asection through the optical plane is shown. Each half of thecell has a 0-5cm. light-path. The cell is 4-9 cm. high andthe central division is 4-0cm. high. All the silica windowsare 1 0mm. thick.

' split' cells, specially made for one ofthe authors by UnicamInstruments Ltd., Cambridge. The cell is shown in Fig. 1.After determination of the spectrum with the two com-ponents in the separate compartments, they were mixed bycovering and inverting the cell and the spectrum of themixture was determined.The polygalacturonic acid was made up in 0 1 M-sodium

phosphate buffer, pH6-5, to give a final concentration of1.0% (wfv) and the Ruthenium Red was made up to aconcentration of 0-2mm in the same buffer. The pH valuesof both components were adjusted to the required value with0-5m-HCI or 0 5m-NaOH immediately before determina-tions. Controls in which an equivalent amount of NaClreplaced the acid or alkali were also performed.

Studie of O.R.D.* Measurements of the O.R.D. ofsolutions containing 2m-moles of galacturonyl residues/I.in 0-2M-sodium phosphate buffer, pH6-5, were determinedby the use of a Bellingham and Stanley Polarmatic 52spectropolarimeter. The apparatus was purged with N2 andmeasurements were made in a 1 0cm. sample cell. For studiesof the effect ofpH on the O.R.D. of polygalacturonic acid,the pH of the samples was adjusted with HCI immediatelybefore the determinations and was checked immediatelyafter. Controls in which HCI was replaced by NaCl were alsorun.The O.R.D. results in the wavelength range 500-250nm.

were interpreted in terms of the Drude equation (see Urnes& Doty, 1961):

aA = A/(A2-A2)

* Abbreviation: O.R.D., optical rotatory dispersion.

864 1969

SOLUTION PROPERTIES OF POLYGALACTURONIC ACIDwhere aA is the specific rotation at a wavelength A and A andAc are constants. The constants A and Ac were determinedfrom the slopes ofplots ofaA A2 against aoA.

RESULTS

Characterization of the polygalacturonic acid. Thespecimen ofpolygalacturonic acidusedwassparinglysoluble in water and readily soluble in dilute alkaliand phosphate buffer. It showed a specific opticalrotation [a]'O + 172.50. Hydrolysis of the material(12mg.) with sulphuric acid, followed by neutraliza-tion and chromatography of the hydrolysate, failedto show the presence of any neutral sugars andyielded only weakly staining spots, that corres-ponded in position and colour reaction to galactu-ronic acid and its oligomers, near the origin.Zone electrophoresis of the polysaccharide in

pyridine-acetic acid buffer, pH 6 5, yielded a singlezone of high mobility towards the anode. The zonewas stained violet by the sulphonated 1-naphtholreagent. Treatment of a sample of the polygalac-turonic acid (4mg.) with pectin pectyl hydrolasegave a product of the same electrophoretic mobilityas the starting material at pH 6 5. A sample of thepolysaccharide (10mg.) was treated with an excessof ethylene oxide and gave a product of greatlydecreased electrophoretic mobility.Zone electrophoresis of the polygalacturonic acid

in tetraborate buffer, pH992, yielded a single zone,which was stained violet by sulphonated 1-naphthol.A portion of the polysaccharide that had been

treated with ethylene oxide (as described above)was further treated with an excess of potassiumborohydride, and the product was recovered andhydrolysed with sulphuric acid. The hydrolysatewas neutralized and analysed by chromatography.In addition to spots corresponding to oligosac-charides, a spot was seen that corresponded togalactose in position and colour reaction with anilinephthalate.The value obtained for the partial specific volume

was 0.65ml./g. The total ash and moisture contentwas below 0.2% by weight.The titration of polygalacturonic acid gave a

simple titration curve with a pK value of 3 7(Fig. 2). Difference titration curves calculated formixtures of polygalacturonic acid with RutheniumRed at a final concentration of 1 mm and withphosphate buffer, final concentration 0-2M, at aninitial pH of 6*5 gave similar results.

Studie8 of the binding of Ruthenium Red. Whensolutions of polygalacturonic acid and RutheniumRed at pH6-5 were mixed, the extinction of thedye increased and the extinction maximum shiftedto a higher wavelength. A similar effect wasproduced when Ruthenium Red was mixed with3M-lithium chloride (Fig. 3). Glycerol in aqueous

28

e 0-10

z 0o05

0

3 4 5 6

pH

Fig. 2. Titration curve of a 0-5% (w/v) solution of poly-galacturonicaciddeterminedat 18°. A2-5ml. sampleofthepolygalacturonic acid was titrated in a water-jacketedvessel with 1OM-NaOH.

E

500 550Wavelength (nm.)

Fig. 3. Spectral shifts accompanying the mixing ofRuthenium Red with polygalacturonic acid and with LiCl.The spectra before and after mixing were recorded in aUnicam SP. 800 recording spectrophotometer at 25' andpH6-5 by using 'split' cells (as illustrated in Fig. 1). Thespectrum of 0-2mM-Ruthenium Red is shown beforemixing ( ), after mixing with 1-0% (w/v) poly-galacturonic acid (----) and after mixing with 6M-LiCI(. . . ).

solutions at concentrations of up to 25% (by vol.)had no effect on the absorption spectrum ofRuthen-ium Red. This metachromic effect that occurredon mixture of the dye with polygalacturonic acidwas markedly dependent on pH. The shift steadilydecreased towards zero with decreasing pH. Thehyperchromic effect also decreased with decreasingwavelength and eventually became a hypochromiceffect with respect to the free dye. The influencesof pH on these shifts are shown in Fig. 4.

Studiem of O.R.D. The O.R.D. curves obtainedfrom polygalacturonic acid between pH2-8 and

Bioch. 1969, 114

Vol. 114 865

R. W. STODDART, I. P. C. SPIRES AND K. F. TIPTON

0o1 12

4

03 4 5 6 7

pH

Fig. 4. Effect of pH on the spectral shifts that occur whenRuthenium Red is mixed with polygalacturonic acid. Themethods used are given in the legend to Fig. 3. The pH ofeach component was adjusted to the required value with0-5M-HCI immediately before the spectra were determined.O, Change in position of absorption maximum after mixing;*, change in value of extinction after mixing.

180 -

50 'X

Ca

o

Ca200 2

co

50

200 250 300Wavelength (nm.)

Fig. 6. O.R.D. of polygalacturonic acid. Details of theprocedures used are given in the legend to Fig. 5.Dispersion at pH3-0; ----, dispersion at pH6-5.

2-4

2-0

1< 1751-1*6

3 4 5 6

pHFig. 5. Variation of the O.R.D. Drude constant, A,, ofpolygalacturonic acid with pH. ThepH ofa polygalacturonicacid solution containing 2m-moles ofgalacturonyl residue/I.was adjusted to the required value immediately before thedetermination of the O.R.D. curve between the wave-lengths 500 and 250nm.

3 4 5 6

pH

Fig. 7. Effect of pH on the sedimentation coefficient ofpolygalacturonic acid. A solution of 1-0% (w/v) poly-galacturonic acid was made up in 0-2 M-potassium phosphatebuffer, pH6-5, and thepH was adjusted to the required valueimmediately before centrifugation. Centrifugation wasperformed at 20°.

6-5 were plane-positive between 500 and 250nm.and yielded linear Drude plots. The variation of theDrude constant Ac with pH is shown in Fig. 5.

Studies of the optical rotation in the far u.v.

showed a peak at about 211nm. at pH6-5, whichmay correspond to the peak of a positive Cottoneffect. At lower pH values the maximum of thispeak shifted to 217nm. (Fig. 6). (Methyl oc-D-galactopyranosid)uronic acid showed a similar peaknear 215nm.; the position of this was not found tovary with pH in the range pH3-3-7 0.

Effects ofpH and salt concentration on the sedimen-tation of polygalacturonic acid. The sedimentationcoefficient of polygalacturonic acid decreased withincreasing pH between pH3-0 and pH6-5 (Fig. 7).

There was no evidence of formation of multiplepeaks at lowpH values as might have been expectedif polymerization had occurred. Plots of sedimenta-tion coefficient versus concentration determined atpH6-5 and pH3-2 were linear between 1-0% and0.2% (w/v) polygalacturonic acid (Fig. 8). Thelower concentration represents the limit of detec-tion of the schlieren optical system used. Thesedimentation coefficient in 0-25M-potassium chlor-ide for a 1.0% (w/v) solution of polygalacturonicacid was 1-8s, close to the value determined atpH 3-8 in the absence of potassium chloride.

Visco8ity of solutions of polygalacturonic acid. Atlow applied pressures the flow of the solutions ofpolygalacturonic acid was markedly non-Newtonian, but at higher pressures (above 15mm.

866 1969

I I

0CQ2Q

SOLUTION PROPERTIES OF POLYGALACTURONIC ACID

2-8

2-4

1-6 - o

I I0 0-5

Conen. (g./100ml.)

Fig. 8. Effect of concentration on thepolygalacturonic acid. The conditions uslegend to Fig. 7. The effects were studiedat pH3-2 (-).

296

2*2

I*8

I*0

0-6

0-2

0 05 1-0

Concn. (g./100ml.)

Fig. 9. Dependence of relative specificin polygalacturonic acid on concentrati0.1M-potassium phosphate buffer, pH6-5

to 2.0% (w/v) in 0 lM-potassium phosphate buffer,pH 6*5, and from these a plot of Thsp./c against c wasderived (Fig. 9), where 8p. is the specific viscosityand c the concentration of polygalacturonic acid(Huggins, 1942). The relative specific viscosity roserapidly at concentrations below 0.5% (w/v) poly-galacturonic acid.

Since the capillary was long and of fine bore andthe volume rate of flow was low, the kinetic correc-tions were far below the limits of experimental errorand were not applied. Likewise the correction forend effects was negligible (Yang, 1961), as weresurface-tension and drainage errors.

Molecular weight of the polygalacturonic acid. Theapparent molecular weight of the polygalacturonicacid was calculated, from the sedimentation andviscosity data by using the equation of Scheraga &Mandelkern (1953) and assuming a value of 2-5 x106 for the constant ,, to be 31600.

DISCUSSION1*0

The sample of polygalacturonic acid used in thesesedimentation of studies was essentially ash- and moisture-free anded are given in the also free of detectable neutral sugars, although veryat pH6-5 (o) and small traces of rhamnose could have escaped

detection because of the resistance of polyuronidechains to acid hydrolysis (Smidsr0d, Haug & Larsen,1966). It was concluded that the polysaccharidewasfree from neutral branches, unlike some nativepectinic acids (Barrett & Northcote, 1965; Stoddartet al. 1967), and was present as the free acid and notas a salt. The high electrophoretic mobility atpH 6 5 and the failure of pectin pectyl hydrolase toincrease the mobility further supported the manu-facturers' claim that the polysaccharide had a verylow ester content. It also indicated again that

0 polysaccharides containing neutral 'blocks' wereabsent. The decrease in electrophoretic mobilityof the polysaccharide after treatment with ethyleneoxide indicated the presence of free carboxylgroups. Reduction of the partially esterified acidfollowed by hydrolysis gave only galactose, againshowing the lack of neutral sugars in the materialand the probable absence of uronic acids other thangalacturonic acid. Electrophoresis of the poly-galacturonic acid in borate buffer gave only a single

I-5 2-0 narrow zone indicative of a high degree of con-figurational homogeneity of the borate complex ofthe polymer (Northcote, 1954). On analytical

vis(osity (iXsp./C) ultracentrifugation the polysaccharide showed aion (c) at 180 of

single peak, which sedimented slowly. Though the*____________ polymer was of heterogeneous molecular weight,

these results show that the distribution of effectiveparent viscosity particle weights about the mean was fairly narrow.ty under condi- The titration curve of polygalacturonic acidlined for various showed a pK value of 3 7, which is close to the valueacid from 0-025 reported by Chowdhury & Neale (1963) for the pK

Hg) the shear-dependence of the apldisappeared. Values of the viscosittions ofNewtonian flow were determconcentrations of polygalacturonic

Vol. 114 867

c:

R. W. STODDART, I. P. C. SPIRES AND K. F. TIPTONof galacturonic acid. A similar value was obtainedfrom difference titrations in the presence of phos-phate buffer and Ruthenium Red.

Shifts in the absorption spectra of dyes have fre-quently been observed when the dyes are bound topolysaccharides. Pal & Basu (1958) and Pal (1958)have interpreted the metachromic shifts that occur

when Methylene Blue or Toluidine Blue is mixedwith acidic polysaccharides as arising from a super-

position of neighbouring dye molecules caused bythe coiling of the polymer chain. Stone (1967) hasalso concluded that metachromacy results, in sub-stantial part, from dye-dye interactions. Considera-tion of the splitting of energy levels of electronssuggests that the enhanced absorption and red shiftobserved in this system represent a J-aggregateand one possible structure of such an aggregate-is-ahelix (Mason, 1964), but Scheibe, Wortz, Haimerl,Seiffert&Winkler (1968) have shown that such spec-

tral shifts can also arise from the adsorption of dyeson surfaces onwhich a helical structure is impossible.With the Ruthenium Red-polygalacturonic acidsystem there appears to be no need to postulate anorderedbindingonthe dye, since the observed shiftscan be simulated by high concentrations of salt.The concentration of lithium chloride required toproduce effects of similar size to those observedwith Ruthenium Red was 3M, which is considerablygreater than the concentration of free carboxylgroups in the solution (about 60mM). This dis-crepancy probably originates from a binding of theRuthenium Red to the polygalacturonic acid in a

highly polar enviroument.Both the spectral shift and the hyperchromic

effect, caused by the interaction of Ruthenium Redwith polygalacturonic acid, were dependent on pHand yielded titration curves (Fig. 4) that resembledthe acid-base titration curve of the polysaccharide.Although the spectral shift decreased towards zero

as the pH was lowered, the hyperchromic effect gaveway to hypochromicity at low pH values. Theorigin of this hypochromic effect is uncertain, but itsuggests that the Ruthenium Red can still interactwith the polygalacturonic acid after the carboxylgroups of the latter have been protonated. Thefailure of glycerol to cause hypochromism in thespectrum of free Ruthenium Red, at concentrationsof glycerol up to 25% by volume, makes it unlikelythat the observed hypochromicity with poly-galacturonic acid arises from a trapping of themolecules of dye in a non-polar enviroument conse-

quent on a collapse of the polymer molecule causedby neutralization of its carboxyl groups.The increase in the Drude constant, Ac, as the pH

islowered gives a titration curve similar to that ofthecarboxyl groups in polygalacturonic acid. Rao &Foster (1963) observed a sharp fall in the specificrotation of amylose as the pH was increased from

11 to 12. This they interpreted as a transformationof the molecule from an imperfect helical form to arandom coil as the pH was raised. In further studieson the effect of pH on amylose, however, Rao &Foster (1965a) interpreted changes in the proton-magnetic-resonance spectra as indicating that thechanges in optical rotation at high pH values maybe due to an increase in the rotational freedomabout the glycosidic bond. With carboxymethyl-amylose, Rao & Foster (1965b) found that both theDrude constant and the specific rotation decreasedwith decreasing pH in the region of the ionization ofthe carboxyl groups and also decreased as the pHwas raised from 11 to 12. If it is assumed that theionized carboxyl groups prevent the formation ofany ordered structures, these results are difficult toreconcile with the helix-coil transition inferred fromthe results with amylose. With polygalacturonicacid the pH-dependent changes in the, Drudeconstant can probably be attributed to the changein the position of the peak of optical rotation in theu.v. Stone (1965) observed a peak of 'opticalrotation at 198nm. with heparin and a trough at188nm. with chondroitin sulphate. Pace, Tanford &Davidson (1964) and Listowsky, Avigad & Englard(1965) have observed a peak in the O.R.D. ofgalactose and its derivatives in the region of 210nm.This peak of optical rotation has been interpreted(Listowsky et al. 1965) as representing a change ofdirection of rotation caused by interaction betweenthe substituent on C-5 of the sugar and the ringoxygen atom, giving rise to a negative Cottoneffect near 190nm. in the C-1 conformation of thegalactose monomers. If this explanation is correctthe shift in position ofthe point ofchange in directionof the optical rotation for polygalacturonic acidcould be caused by either an increase in the propor-tion of the residues in the C-1 conformation as thepH was lowered, or some further, unidentified,interactions. Hirano, Manabe, Miyazaki & Onodera(1968) have shown that the sugar residues inacetylated polygalacturonic acid are mainly in theC- 1 conformation. An increase in the proportion ofsugar residues in the C-1 conformation in poly-galacturonic acid as thepH is loweredmay reflect theformation of some ordered structure in whichinteraction of sugar residues causes a shift in theirconformational equilibria.The value of 1-83 calculated (at pH values above

5.0) for the sedimentation coefficient, S'20, forpolygalacturonic acid of this range of molecularweights indicates that the molecules have a highdegree ofasymmetry.The sedimentation of polygalacturonic acid was

found to be strongly dependent on the pH (Fig. 7).The increases in the sedimentation coefficient as thepH was lowered resembled a titration curve with apoint of inflexion near to that of the acid-base

868 1969

Vol. 114 SOLUTION PROPERTIES OF POLYGALACTURONIC ACID 869titration curve of polygalacturonic acid. Thisincrease of sedimentation coefficient with loweringof the pH could arise from the molecule's becomingmore compact as the carboxyl groups becameprotonated, or from aggregation. Plots of sedi-mentation coefficient against concentration werelinear both at pH 341 and at pH 6-5 (Fig. 8), andgave no sign of the curve that would be expectedfrom an aggregation dependent on concentration.Thesedimentationcoefficientofthepolygalacturonicacid at pH 6-5 was increased by the presence of 0-25M-potassium chloride, which is consistent with thesuggestion that the neutralization of the charge ofthe carboxyl groups leads to the dependence of thesedimentation coefficient on pH.

Studies of the viscosity of solutions of poly-galacturonic acid showed anomalies both in thedependence of the viscosity on the rate of shear andin the dependence ofthe relative specific viscosity onconcentration. The former, non-Newtonian, be-haviour could be explained as a charge-repulsioneffect between the macroions of the polymer, as anorientation effect of asymmetric molecules or as aneffect of the interaction of hydration shells of thepolymer. Although all three effects are likely to beinvolved to some extent the first of them (the'second electroviscous effect'; Yang, 1961) shouldbe much decreased at high dilution and in the pre-sence of salt; under these conditions the non-Newtonian behaviour was still observed. Possibleinteractions ofhydration shells would also disappearat sufficiently high dilution, and an orientationeffect is the most probable explanation.The value of the partial specific volume was

closely similar to those previously reported forneutral polysaccharides (Bell, Gutfreund, Cecil &Ogston, 1948; Olaitan & Northcote, 1962) and theglycopeptide of yeast cell walls (Korn & Northcote,1960; Sentandreu & Northcote, 1968). It thereforeseems unlikely that the hydration of poly-galacturonic acid in solution is substantiallydifferent from that of neutral polysaccharides.The first part of the relative specific viscosity-

concentration curve shows a linear relationship,which yields a value of the intrinsic viscosity [X] of0.48ml./g. on extrapolation and a value of 5-4 forthe Huggins constant (Huggins, 1942). Thisintrinsic viscosity corresponded to that given by aprolate ellipsoid of rotation of axial ratio 26, ascalculated from the equation of Simha (1940) andMehl, Oncley& Simha (1940) assumingno hydration.Ifa 30% (w/v) hydration is assumed the axial ratio isdecreased to 21, as calculated from the equations ofOncley (1941). Both values of the axial ratio arevery small for a linear polymer ofas high a molecularweight as that of polygalacturonic acid. This isexplicable ifthe molecule isflexible, is folded back onitself or is associated to give a rounded or branched

aggregate. The very high value of the Hugginsconstant is probably caused by the polyionic natureof the molecule and is about ten times larger than atypical value for a neutral flexible polymer. Inview of the non-Newtonian behaviour of thesolutions the validity of applying the Hugginsequation is uncertain and the significance of thevalue ofthe Huggins constant is rather doubtful. Itis also questionable whether the extrapolation todetermine the intrinsic viscosity is valid for theanomalous plots ofFig. 9.The increase of the relative specific viscosity at

low concentrations may be considered to representan increase in the radius of gyration of the effectivehydrodynamic particle equivalent to its becoming anellipsoid of rotation of axial ratio greater than 50.Such an increase could result from increased rigidityof a flexible molecule, an expansion of a moleculecoiled on itself, a large change in hydration, adissociation of a rounded or branched aggregateinto asymmetric subunits or the extension of anasymmetric molecule. It is not possible to distinguishbetween these, although a sufficiently large changein hydration is unlikely to occur.The model of polygalacturonic acid in solution

that emerges from these studies is that of anextended molecule with some flexibility, which ispartially maintained in its conformation by therepulsion of charges on the carboxyl groups ofadjacent residues. Consequently effective neutral-ization of these charges at low pH values or highionic strength leads to a collapse of the molecule,with possible attendant changes in the conformationof the residues. It seems likely that the hydroxylgroups of polygalacturonic acid play an importantpart in determining the structure of the molecule,especially under conditions in which charge-repulsion effects are minimal. Hence any futureconsiderations of the role of galacturonans in theplant cell wall must take account, not only of thedegree of esterification of the carboxyl groups, butalso of the distribution of ester bonds, the degree ofsubstitution of hydroxyl groups and the ways inwhich ions such as Ca2+ may interact with hydroxylas well as carboxyl groups. The situation in vivowill be further complicated by the presence ofneutral sugars and branches in the galacturonans,but their general behaviour is likely to approximateto that described here.

We thank Dr D. H. Northcote, F.R.S., for his advice.

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