Thermally Induced Conformation Change of Succinoglycan inAqueous Sodium Chloride

Tomoko Nakanishi and Takashi Norisuye*

Department of Macromolecular Science, Graduate School of Science, Osaka University,Toyonaka, Osaka 560-0043, Japan

Received December 4, 2002; Revised Manuscript Received January 27, 2003

Static and dynamic light scattering, viscosity, and optical rotation measurements have been made at eightdifferent temperatures between 25 and 75°C on two succinoglycan samples (sodium salt) with weight-average molecular weightsMw of 7.14× 105 and 3.54× 105 (at 25°C) in 0.01 M aqueous NaCl to investigatethe thermally induced order-disorder conformation change of the polysaccharide. Additionally, viscometryand polarimetry have been performed for a sodium salt sample (Mw ) 4.55 × 105 at 25 °C) whoseMw,z-average radius of gyration⟨S2⟩z

1/2, and hydrodynamic radiusRH in the aqueous salt had been determinedpreviously. As the temperature increases,Mw, ⟨S2⟩z

1/2, RH, and the intrinsic viscosity for every sample sharplydecrease around 55°C where the specific rotation at 300 nm sigmoidally increases. In particular,Mw at 25°C (i.e., in the ordered helical state) is twice as large as that at 75°C (i.e., in the disordered state). Thesefindings substantiate that the ordered structure is composed of two chains and hence is a double helix. Dataanalysis shows that this helix at 25°C is characterized by an unperturbed wormlike chain with a helix pitchof about 2 nm (per repeating unit) and a persistence length of about 50 nm and that upon heating, it dissociatesdirectly (i.e., in all-or-none fashion) to disordered chains of a similar contour length but with a much smallerpersistence length of about 10 nm. The temperature dependence of the light scattering second virial coefficientis discussed in relation to the association of disordered chains in the cooling process.

Introduction

Succinoglycan is an ionic polysaccharide produced by suchbacterial species asPseudomonas, Rhizobium, Agrobacte-rium, and Alcaligenes.1 Its repeating unit consists of fourmain-chain residues and a tetrasaccharide side chain with asuccinate monoester and a pyruvate (Figure 1), but thecontent of the succinate monoester varies depending onbacterial source or cultivation condition.1-3 As was shownby optical rotation,4-6 circular dichroism,4 and othertechniques,5,7-10 the polysaccharide in aqueous solution withor without added salt assumes an ordered helical conforma-tion at low temperatures and undergoes a conformationchange to a disordered state with raising temperatureT. Thehelical structure is, however, a matter of debate, in thatmeasured solution properties, often suggestive of intermo-lecular association,4,7,9,11-13 led several groups4-6,9,13,14 topropose a single or double helix on the basis of differentpieces of experimental evidence. The crystalline structureof succinoglycan is unknown probably because of too poorcrystallinity for X-ray diffraction. Thus, experimental de-termination of the ordered polysaccharide structure is achallenging problem of solution workers.

In recent work,6 we found from analyses ofz-average radiiof gyration⟨S2⟩z

1/2 and intrinsic viscosities [η] that succino-glycan in 0.1 M aqueous NaCl at 25°C, i.e., in the orderedstate, behaves as a semirigid chain with a linear mass densityalmost twice as large as that expected for the single chainand concluded that its predominant molecular species presentin the aqueous salt is a double helix or a lateral aggregate

composed of paired single helices. Evidently, both modelsrequire the molecular weight to be halved when the polymeris brought to the completely disordered state at an elevatedtemperature. More recently,15 we showed this to be indeedthe case for a sample (designated S-5) in 0.01 M aqueousNaCl by light scattering. Furthermore, we found that⟨S2⟩z

1/2

and the hydrodynamic radiusRH for this sample sharplydecrease with increasingT around 55°C where the substan-tial decrease in weight-average molecular weight,Mw, takesplace. Kaneda et al.14 also reported thatMw in 0.1 M aqueousNaCl at 75°C was roughly half that at 25°C. Since thesefindings are necessary conditions for both of the double-helix and aggregate models, a detailed analysis of thermallyinduced changes in chiroptical and conformation-dependentproperties is mandatory in order to distinguish between thetwo models. No such work has as yet been reported.

In the present study, we made static and dynamic lightscattering, viscosity, and optical rotation measurements on

Figure 1. Repeating unit of succinoglycan (sodium salt). A succinatemonoester may be linked to either one or both of the two side-chainglucose residues.

736 Biomacromolecules 2003,4, 736-742

10.1021/bm020132f CCC: $25.00 © 2003 American Chemical SocietyPublished on Web 03/21/2003

two Na salt samples of succinoglycan in 0.01 M aqueousNaCl at different temperatures between 25 and 75°C toclarify the order-disorder change of the polysaccharide andto determine the ordered structure. We also carried outpolarimetry and viscometry for sample S-5 whoseMw, ⟨S2⟩z,andRH had already been determined as functions ofT.15 Thedata obtained forMw, ⟨S2⟩z, RH, [η], and the specific rotationare presented and analyzed below.

Experimental Section

Samples.Previously prepared Na salt samples,6 S-4 andS-5, and a newly prepared sample (designated below as S-9)were used for the present work. Their original material wasRhone-Poulenc Rheozan (fromAgrobacterium tumefaciens)with a D-glucose/D-galactose/pyruvate/succinate ratio of7:1:1:0.8. Sample S-9, a middle fraction, was prepared inthe following way from a sonicated sample (1.3 g, [η] )4.6× 102 g cm-3 in 0.01 M aqueous NaCl at 25°C), whichhad been prepared previously by 20-kHz sonic irradiationfor 28 h at 0°C followed by purification (see ref 6). Thesonicated sample was divided into three parts by fractionalprecipitation at 25°C with 0.5 M aqueous sodium acetateas the solvent and methanol as the precipitant, and then themiddle portion (0.8 g) was further fractionated into three.The central fraction S-9 (0.5 g) thus obtained was repre-cipitated from a 0.5 M aqueous sodium acetate solution intomethanol, washed with methanol three times and then withacetone, and vacuum-dried. After its aqueous solution (1%)had been passed through a mixed-bed ion exchanger(Amberlite IR-120 + IR-400), the acid succinoglycansolution (pH∼2.8) was neutralized with 0.05 N aqueousNaOH (Beckmanφ 70 PH meter) at a temperature below25 °C and lyophilized over more than 4 days.

Each sample, further dried in a vacuum at room temper-ature for 1 day, was mixed with 0.01 M aqueous NaCl atabout 25°C to prepare test solutions. The polymer massconcentrationc was calculated from the gravimetricallydetermined weight fraction with the solvent density.

Static Light Scattering. Scattering intensity data weretaken for samples S-4 and S-9 at 25-75 °C on a Fica-50light scattering photometer in an angular range from 30 to150°, using vertically polarized incident light of wavelength(λ0) 436 or 546 nm. Five solutions of different polymerconcentrations were subjected to alternate measurements ofelastically and quasi-elastically scattered intensities in theorder of raisingT (see below for dynamic light scattering).Virtually no time dependence of scattered intensity wasobserved after each solution had been thermally equilibrated.It should be noted that the thermally induced conformationchange of the polysaccharide is not always reversible asdetected by optical rotation4-6 and relative-viscosity4-8

measurements.Calibration of the apparatus and optical clarification of

test solutions were carried out as described previously.6

Necessary values for the specific refractive index incrementof Na salt succinoglycan in 0.01 M aqueous NaCl at fixedchemical potentials of diffusible components were obtainedby interpolation or extrapolation of the previously data:15

0.146, 0.144, and 0.141 cm3 g-1 at 25, 45, and 65°C,respectively, for 436 nm and 0.144, 0.142, and 0.139 cm3

g-1 at 25, 45, and 65°C, respectively, for 546 nm.

As in our previous work,6,15 Mw and A2LS (the light

scattering second virial coefficient) were evaluated by useof the (Kc/Rθ)1/2 vs c plot, while ⟨S2⟩z was determined bythe Kc/Rθ vs k2 plot. Here,K is the optical constant,Rθ isthe excess reduced scattering intensity at scattering angleθ,andk is the magnitude of the scattering vector defined byk) (4πn0/λ0) sin(θ/2), with n0 being the solvent refractiveindex. TheMw values for samples S-4 and S-5 at 25°Cagreed with the previously determined values6 in 0.1 Maqueous NaCl at 25°C within (3%. In Figure 2 of ourprevious paper (ref 15), the graph of (Kc/Rθ)c)0 ()Kc/Rθ atinfinite dilution) vs k2 for sample S-5 was erroneouslyprinted. Since this graph is misleading, we show the correctone in Figure 2 of the present paper. The plots for samplesS-4 and S-9, with which the present experiment wasconcerned, were similar to those in this figure.

Dynamic Light Scattering. Normalized autocorrelationfunctionsg(2)(t) at timet were obtained for 0.01 M aqueousNaCl solutions of samples S-4 and S-9 at scattering anglesbetween 30 and 90°, using an ALV/DLS/SLS light scatteringphotometer equipped with an ALV-5000E WIN correlatorand a 532 nm YAG laser. The experimental first cumulantΓ was evaluated from the slope of a plot of ln[g(2)(t) - 1]againstt at a fixedθ (or k) for each solution according tothe relation ln[g(2)(t) - 1] ) -2Γt + constant. The plotswere linear fork2t below (1-2) × 106 cm-2 s and yieldedΓ/k2 essentially independent ofk for all solutions. The valuesof (Γ/k2)k)0, i.e., the zero-angle values ofΓ/k2, at a givenTwere extrapolated to infinite dilution to obtain the transla-tional diffusion coefficientD. The concentration dependenceof (Γ/k2)k)0 for samples S-4 and S-9 was similar to whatwas shown previously for sample S-5 in Figure 6 of ref 15;

Figure 2. Corrected graph of (Kc/Rθ)c)0 vs k2 for succinoglycansample S-5 in 0.01 M aqueous NaCl at different temperatures (theoriginal graph appearing in Figure 2 of ref 15 is incorrect). The ordinatevalues at the respective temperatures are shifted by A as indicated.

Succinoglycan Conformation Change Biomacromolecules, Vol. 4, No. 3, 2003 737

the ordinate values and the ordinate variable in this figurewere also erroneously printed, but their correction is notgiven here.

Viscometry. Four solutions of different polymer concen-trations were prepared for samples S-4, S-5, and S-9, andtheir zero-shear rate viscosities were determined at 25-75°C (in the order of raisingT) using a low-shear four-bulbviscometer of the Ubbelohde type with apparent shear ratesof 13-91 s-1. Huggins’ constants were generally normal(between 0.39 and 0.49), though they exceptionally exceeded0.5 in a few measurements at high temperatures.

Polarimetry. Specific rotations [R]300 atλ0 ) 300 nm weremeasured for samples S-4, S-5, and S-9 in 0.01 M aqueousNaCl at 25-70 °C on a Jasco J-725 CD spectropolarimeterequipped with an ORD detector. A cylindrical quartz cell of10 cm path length was used. The polymer concentration wasadjusted to about 0.3 wt %.

Figure 3 shows the temperature dependence of [R]300

determined for the three samples withT successively raised.The sigmoidal rises of the curves confirm the earlier finding4,6

that succinoglycan in 0.01 M aqueous NaCl undergoes anorder-disorder conformation change around 55°C. Thischange slightly shifts to a higherT for a higher molecularweight (see Table 1 forMw), but for any sample the orderedand disordered conformations are predominant below 40°Cand above 65°C, respectively.

Results and Discussion

Changes in Molecular Weight and Chain Dimensions.Numerical results from light scattering and viscometry in0.01 M aqueous NaCl are summarized in Table 1, in whichthe data ofMw, A2

LS, ⟨S2⟩z1/2, andD for sample S-5 are the

reproductions from our previous paper.15 As T increases from25 to 75 °C, Mw for any sample decreases to one-half,confirming the previous finding15 (on sample S-5) that theordered helical structure in the aqueous salt is composed oftwo chains. With this increase inT, ⟨S2⟩z

1/2 and [η] alsodecrease, whileD increases. Hence, the order-disorderchange is accompanied not only by the dissociation of paired

chains but also by a decrease in molecular dimensions. Thevalues ofA2

LS in the table are on the order of 10-3 mol g-2

cm3 regardless ofT. The strong repulsion between helices(in the ordered state) as indicated by this largeA2

LS doesnot seem to support the aggregate model mentioned in theIntroduction. Interestingly,A2

LS has a small minimum around55 °C in common with the three samples. In the followingpresentation, we use the hydrodynamic radius defined byRH

) kBT/6πη0D, instead ofD, sinceD contains the effects ofT and solvent viscosityη0 (kB denotes the Boltzmannconstant).

Figures 4 through 7 show the temperature dependence ofMw, ⟨S2⟩z

1/2, RH, and [η] for the three samples. The indicatedcurves are explained in the next subsection. Importantly, thesubstantial decrease in these quantities (with increasingT)occurs in theT range between 45 and 60°C in which [R]300

increases sigmoidally (Figure 3). This finding cannot bereconciled with the lateral aggregate model unless dissocia-tion of “aggregated single-helices” happens to be ac-companied by a cooperative helix-disordered chain transition.Hence, it is legitimate to conclude that the ordered confor-mation of succinoglycan in 0.01 M aqueous NaCl is a double-helical dimer.

Order-Disorder Change. The data in Figures 3-7suggest that the dissociation of the succinoglycan dimerproceeds not through partial breaking of helical portions butdirectly to single chains. If only intact double helices (species2) and disordered single chains (species 1) coexist in the

Figure 3. Temperature dependence of [R]300 for succinoglycansamples S-4, S-5, and S-9 in 0.01 M aqueous NaCl.

Table 1. Results from Light Scattering and ViscosityMeasurements on Succinoglycan Samples in 0.01 M AqueousNaCl at Different Temperatures

T, °C Mw × 10-5

A2LS × 103,

mol g-2 cm3 ⟨S2⟩z1/2, nm

D × 107,cm2 s-1

[η] × 10-2,cm3 g-1

sample S-425 7.14 1.01 101 0.740 11.235 7.04 1.04 100 0.960 10.945 6.58 1.04 99.0 1.22 10.750 6.10 1.03 94.1 1.38 10.555 5.59 0.96 89.1 1.58 10.160 4.76 0.94 67.8 1.96 8.9565 3.72 1.16 46.8 2.37 3.4575 3.59 1.17 44.7 2.78 3.31

sample S-525 4.55a 1.52a 68.5a 0.960a 6.7035 4.50a 1.62a 67.9a 1.23a 6.5545 4.37a 1.52a 66.7a 1.54a 6.4550 4.08a 1.38a 61.2a 1.72a 6.4055 3.70a 1.32a 56.6a 1.96a 6.0060 3.03a 1.67a 48.5a 2.30a 3.2965 2.33a 1.69a 34.3a 2.85a 2.5275 2.27a 1.65a 32.4a 3.40a 2.50

sample S-925 3.54 1.90 55.5 1.02 4.9235 3.33 1.95 55.4 1.31 4.8545 3.17 1.74 52.1 1.65 4.5450 2.97 1.75 50.2 1.92 4.4255 2.78 1.74 45.9 2.38 4.0560 2.27 2.02 40.9 2.80 2.2265 2.03 2.36 31.3 3.20 1.9275 1.94 2.36 30.7 3.80 1.91

a Taken from ref 15.

738 Biomacromolecules, Vol. 4, No. 3, 2003 Nakanishi and Norisuye

solution at a givenT, the measured [R]300 ()[R]), Mw, ⟨S2⟩z1/2,

RH, and [η] may be expressed by

Here, f denotes the weight fraction of double helices andthe subscripts 1 and 2 signify the species 1 and 2, respectively(M2, for example, is the molecular weight of the doublehelix). It should be noted thatD from dynamic light scatteringis z-averaged (as is the case with⟨S2⟩z) and that all the aboverelations hold for a polydisperse sample ifMi (i ) 1, 2), forexample, is replaced by the weight-average molecular weightof the speciesi. The quantities⟨S2⟩i, RHi, and [η] i may beweakly decreasing functions ofT due to possible changesin chain flexibility with T, but their temperature dependenceis ignored in the present analysis.

The values off for each sample were evaluated as afunction of T from [R]300 in Figure 3 using eq 1 with [R]2

and [R]1 taken as the [R]300 values at 25 and 75°C,respectively. The curves in Figures 4 through 7 show therespective properties calculated from eqs 2-5 with thosefvalues and the properly chosenMi, ⟨S2⟩i, RHi, and [η] i (i.e.,the values at 25 and 75°C indicated by the curves). Theirsatisfactory fits to the data points substantiate the validityof our assumption that only double helices and single chainscoexist in the aqueous salt. In other words, upon heating,the double-helical dimer of succinoglycan dissociates directly(i.e., in all-or-none fashion) to disordered chains.

When a 0.01 M NaCl solution of single chains is cooledto 25 °C, [R]300 almost recovers the original value for thedouble helix, but the relative viscosity stays halfway.6 It wasfound in this work (after the measurement at 75°C) that,thoughMw for sample S-4 increases on cooling, it stays farbelow the value for the dimer. These observations indicatethat both restoration of the (local) helical conformation andintermolecular association take place. Thus, the interactionbetween single chains should be predominantly attractive atlow T despite the fact that it is strongly repulsive at 75°Cas indicated by theA2

LS data in Table 1. This is plausible iflowering of T enhances attractions between main-chainresidues (belonging to different chains) exposed to theaqueous medium. Evidently, the second virial coefficient for

Figure 4. Temperature dependence of Mw for samples S-4, S-5, andS-9 in 0.01 M aqueous NaCl. The data for S-5 are from ref 15. Curvesare from eq 2 with the f values estimated from the [R]300 data in Figure3.

Figure 5. Temperature dependence of ⟨S2⟩z1/2 for samples S-4, S-5,

and S-9 in 0.01 M aqueous NaCl. The data for S-5 are from ref 15.Curves are from eq 3.

Figure 6. Temperature dependence of RH for samples S-4, S-5, andS-9 in 0.01 M aqueous NaCl. The data for S-5 are from ref 15. Curvesare from eq 4.

Figure 7. Temperature dependence of [η] for samples S-4, S-5, andS-9 in 0.01 M aqueous NaCl. Curves are from eq 5.

[R] ) f[R]2 + (1 - f)[R]1 (1)

Mw ) fM2 + (1 - f)M1 (2)

⟨S2⟩zMw ) fM2⟨S2⟩2 + (1 - f)M1⟨S

2⟩1 (3)

Mw/RH ) fM2/RH2 + (1 - f)M1/RH1 (4)

[η] ) f[η]2 + (1 - f)[η]1 (5)

Succinoglycan Conformation Change Biomacromolecules, Vol. 4, No. 3, 2003 739

the single-chain species is required to be stronglyT-dependent and negative at lowT. In the Appendix, weshow that on some conditions, ourA2

LS data, insensitive toT throughout the entire range studied (see Table 1), satisfythis requirement and hence are consistent with the single-chain association in the cooling process.

Ordered and Disordered Conformations.According toour previous analysis6 of the molecular weight dependenceof ⟨S2⟩z and [η] based on the wormlike chain,16 the orderedhelical conformation of succinoglycan in 0.1 M aqueousNaCl at 25°C is characterized by a linear mass densityML

of 1500 ((170) nm-1 and a persistence lengthq of 50 ((8)nm. In the analysis of [η], the Yamakawa-Fujii-Yoshizakitheory17,18 was used on the assumption that the diameterdis within the range 1.5-3.5 nm, and for⟨S2⟩z, samplepolydispersity6 (the z to weight-average molecular weightratio Mz/Mw ) 1.25-1.3) was taken into account using thefollowing expression19 based on the Schulz-Zimm distribu-tion function withMz/Mw ) 5/4

where

The model parameters for the double helix in 0.01 M aqueousNaCl may hardly differ from the above values in 0.1 Maqueous NaCl, since it was previously6 found that [η] in theformer solvent was only 1-7% larger than that in the latterfor any sample ranging inMw from 1.0× 105 to 8.7× 106.

Figure 8 shows that the previous and present [η] data (theunfilled and filled circles, respectively) in 0.01 M aqueousNaCl at 25°C are fitted closely by the theoretical solid curvefor ML ) 1460 nm-1, q ) 56 nm, andd ) 2.5 nm. The firsttwo parameters contain uncertainties comparable to thoseindicated above for 0.1 M NaCl solutions ifd is chosen asa certain value between 1.5 and 3.5 nm. The present⟨S2⟩z

data are also found to be explained by the wormlike chainwith ML ) 1520 nm-1 and q ) 50 nm (not shown) whenanalyzed according to eq 6 with the aid of the previous data6

at 0.1 M NaCl. Since these parameters are in substantial

agreement with those from [η], we may conclude that thesuccinoglycan double helix in 0.01 M aqueous NaCl ischaracterized byML ) 1500 ((170) nm-1 andq ) 53 ((8)nm.

To deduce the disordered conformation, we additionallymade light scattering and viscosity measurements on Na saltsample S-3 (see ref 6) in 0.01 M aqueous NaCl at 75°C,with the result thatMw ) 9.07× 105, A2 ) 4.4× 10-4 molg-2 cm3, ⟨S2⟩z

1/2 ) 77.1 nm,D ) 1.71× 10-7 cm2 s-1, and[η] ) 6.44× 102 cm3 g-1. This Mw is almost exactly halfthat (1.85× 106) previously6 determined in 0.1 M aqueousNaCl at 25°C. The dashed line in Figure 8 shows that theunperturbed wormlike chain withML ) 860 nm-1, q ) 10nm, andd ) 2 nm explains the [η] data (the triangles) in0.01 M aqueous NaCl at 75°C; if d is taken as 2.0( 0.5nm, close fits are obtained forML ) 860 ((120) nm-1 andq ) 10 ((2.5) nm. Excluded-volume effects on [η] may notbe very significant, if any, because the Kuhn segment number() Mw/2qML) for the highest molecular weight examined isabout 50, below which those effects in typical stiff chainsare negligible.20,21

The data of⟨S2⟩z1/2 andRH for disordered succinoglycan

(at 75°C) were similarly analyzed using eq 6 for the formerand the Yamakawa-Fujii theory22 for the latter. The linesin panels A and B of Figure 9, representing the theoretical⟨S2⟩z

1/2 for ML ) 760 nm-1 andq ) 11 nm and the theoreticalRH for ML ) 740 nm-1, q ) 11 nm, andd ) 2 nm,respectively, come close to the data points. These parametersare only moderately accurate, but they do not differ muchfrom the above estimates from [η]. In short, belowMw ∼ 9× 105, the single chain at 75°C behaves like an unperturbedwormlike chain withML ) 800 ((180) nm-1 andq ) 10((3) nm.

Table 2 summarizes the model parameters for ordered anddisordered succinoglycan in 0.01 M aqueous NaCl. TheML

of 1500 ((170) nm-1 for the former yields 2.0 ((0.2) nmfor the pitchh of the double helix per repeating unit sincethe molar mass of the polysaccharide repeat unit with anaverage succinate content of 0.8 (see the Experimentalsection) is approximately 1490. Thish implies that the double

Figure 8. Data of [η] for succinoglycan in 0.01 M aqueous NaCl at25 °C (circles) and at 75 °C (triangles), compared with the theoreticalcurves for the unperturbed wormlike cylinders17,18 with ML ) 1460nm-1, q ) 56 nm, and d ) 2.5 nm at 25 °C (the solid line) and withML ) 860 nm-1, q ) 10 nm, and d ) 2 nm at 75 °C (the dashedline). The unfilled circles were taken from ref 6.

⟨S2⟩z/q2 ) (5/12)x - 1 + (2/x) - [8/(3x2)] +

[8/(3x2)][1 + (x/4)]-3 (6)

x ) Mw/(qML) (7)

Figure 9. Statistical and hydrodynamic radii (circles) for disorderedsuccinoglycan in 0.01 M aqueous NaCl at 75 °C, compared with thetheoretical curves for unperturbed wormlike chains: line in panel A,⟨S2⟩z

1/2 calculated from eq 6 with ML ) 760 nm-1 and q ) 11 nm; linein panel B, RH calculated from the Yamakawa-Fujii theory22 with ML

) 740 nm-1, q ) 11 nm, and d ) 2 nm.

740 Biomacromolecules, Vol. 4, No. 3, 2003 Nakanishi and Norisuye

helix is considerably extended along the helix axis. Thedisordered chain has anML roughly half that for the doublehelix, so that its contour length per repeating unit iscomparable toh.

The rigidity of the succinoglycan double helix, expressedin terms ofq, is about 50 nm in both 0.01 and 0.1 M NaClsolutions, though the slightly largerq (53 nm) in the formersolvent than the previously estimated value (50 nm) in thelatter may be a reflection of the stiffness effect23-25 ofenhanced electrostatic repulsions between charged groupsat the lower ionic strength. Dentini et al.4 reported that [η]for a succinoglycan sample (Mw ) 3.3 × 106) in aqueousNaCl at 25°C is very insensitive to salt concentration overthe range from 0.01 to 1 M. Hence, ourq of about 50 nmmay be regarded as the intrinsic persistence length of thedouble helix at infinite ionic strength. This value is compa-rable to what is known for double-stranded DNA at highionic strength.26 On the other hand, the single chain ofdisordered succinoglycan at 75°C has a much higherflexibility as indicated by aq of 10 nm. Its stiffness iscomparable to that for cellulose derivatives.27

Conclusions

The chiroptically detected order-disorder conformationchange of succinoglycan in 0.01 M aqueous NaCl isaccompanied by sharp decreases in molecular weight,statistical and hydrodynamic radii, and intrinsic viscosity withraising temperature around 55°C and is a process in whicha double-helical dimer dissociates to single chains notthrough partial breaking of helical portions but directly, i.e.,in all-or-none fashion. The double-helical conformation at25 °C is characterized by a helix pitch of 2.0 ((0.2) nm perrepeating unit and a persistence length of 53 ((8) nm. Thesingle disordered chain at 75°C has a contour lengthcomparable to that of the double helix, but it has a muchhigher flexibility as indicated by a persistence length of about10 nm. Though the measured light scattering second virialcoefficients are positive and large throughout the temperaturerange from 25 to 75°C, the second virial coefficient for apair of single chains can be strongly temperature dependentand negative at low temperatures where succinoglycan chainsonce disordered at 75°C tend to undergo intermolecularassociation in addition to restoration of the (local) helicalconformation.

Appendix

Although A2LS in Table 1 is insensitive toT, it has a

shallow minimum around 55°C in common with the threesuccinoglycan samples and hence beyond experimental error.The presence of such a minimum prompted us to examine

the second virial coefficientA11 for the single-chain speciesin relation to the tendency of intermolecular association inthe cooling process. In this Appendix, we attempt to extractA11 from theA2

LS data by an unsophisticated analysis.The light scattering second virial coefficient for a binary

polymer mixture consisting of species 1 (single chains withmolecular weightM1) and 2 (double helices with molecularweight M2) is expressed by28,29

where f is the weight fraction of double helices (as in thetext) and the second virial coefficientAij for speciesi and jis given by30

with

In these equations,NA is the Avogadro constant,MLi is theML for speciesi whose contour length equals the product ofthe total bead number and the bead spacingai, âij is the binarycluster integral for the interaction between a pair of beadsbelonging to speciesi andj, andhij is the so-calledh functionrepresenting bead-bead interactions higher than the singlecontact;30-32 for polyelectrolytes,âij contains both electro-static and nonelectrostatic contributions. Except for nonionicflexible chains, no expression forh12 is known even in arough approximation.

We equateML1 with ML2/2 for simplicity (see Table 2)and assume thatX12 ) (X11 + X22)/2. These conditions andeq 6 withM2 ) 2M1 allow eq A-1 to be simplified to

If the main-chain residues in the succinoglycan helix aresurrounded by ionic side chains (differing from those in thesingle chain), the second virial coefficientA22 for the double-helix species is determined substantially by hard-core andelectrostatic interactions.33 In this case, we may ignore thetemperature dependence ofA22 in a first approximation, sincethe Debye lengthκ-1 ()(8πQns)-1/2) and the Bjerrum lengthQ ()e2/εKBT) are insensitive toT in the range of our interest,wherens is the number density of added salt (1/1 electrolyte),e the elementary electric charge, andε the dielectric constantof water.

The experimental data ofA2LS/A22 (A22 ) A2

LS at 25°C)for the three succinoglycan samples, plotted againstf, roughlyform a composite curve with a shallow minimum aroundf) 0.5 (not shown) and are fitted approximately by eq A-4when A11/A22 is expressed as 1.16- 1.9f. The theoreticalcurve for each sample can then be converted to theA2

LS/A22

vs T relation using the [R]300 data in Figure 3. Figure 10shows that the calculatedA2

LS/A22 vs T curve quite closelyfitting the data points for sample S-4 reproduces theexperimentally observed shallow minimum and subsequentrise (with an increase inT). The curves for the lowermolecular weight samples (S-5 and S-9) also exhibited these

Table 2. Wormlike-Chain Parameters for Succinoglycan in 0.01 MAqueous NaCl at 25 and 75 °C

T, °C ML, nm-1 q, nm d, nma

25 1500 ((170) 53 ((8) 2.5 ((1)75 800 ((180) 10 ((3) 2.0 ((0.5)

a Assumed.

A2LS ) [f2M2

2A22 + 2f(1 - f)M1M2A12 +

(1 - f)2M12A11]/Mw

2 (A-1)

Aij ) (NA/2MLiMLj)Xij (A-2)

Xij ) (âij/aiaj)hij (A-3)

A2LS/A22 ) (1 + f)-2[4f + (1 - f)(A11/A22)] (A-4)

Succinoglycan Conformation Change Biomacromolecules, Vol. 4, No. 3, 2003 741

features (not shown for clarity) and shifted slightly to a lowerT in accordance with theMw-dependent curve of [R]300 vsT.

The above empirical relation,A11/A22 ) 1.16 - 1.9f,indicates thatA11 vanishes atf ) 0.61, i.e., atT ) 54-57°C. If literally taken, the interaction between single chainsis attractive and repulsive below and above thiscriticaltemperature (about 55°C), respectively. If correction is madefor the T-dependence ofA22 using the current theory foruniformly charged long cylinders in the Debye-Huckelapproximation (see eqs 3, 4, and 6 in ref 33), a differentrelation,A11/A22 ) 1.16- 1.6f, is found to give close fits totheA2

LS/A22 data as in Figure 10. Thecritical T in this caseis 52-54 °C, being only slightly lower than the aboveestimate. In short,A11 changes its sign at aT of about 55°C, i.e., around the midpoint of the order-disorder change,when the conditions or approximations invoked in theanalysis are satisfied. This conclusion seems quite interesting,but not much quantitative significance should be claimedfor the A11/A22 vs f relation and the critical temperaturebecause of the invoked approximation toX12. The point tobe made here is that the measured positive, largeA2

LS valueswith a seemingly trivial minimum conceal the strong,negativeT-dependence ofA11 and do not contradict thetendency of intermolecular association of single succino-glycan chains in the cooling process.

References and Notes

(1) Harada, T.; Harada, A. InPolysaccharides in Medical Applications;Dumitriu, S., Ed.; Marcel Dekker: New York, 1996; p 21.

(2) Harada, T.; Amemura, A.; Jansson, P. E.; Lindberg, B.Carbohydr.Res. 1979, 77, 285.

(3) Matulova, M.; Toffanin, R.; Navarini, L.; Gilli, R.; Paoletti, S.;Cesaro, A.Carbohydr. Res. 1994, 265, 167.

(4) Dentini, M.; Crescenzi, V.; Fidanza, M.; Coviello, T.Macromolecules1989, 22, 954.

(5) Gravanis, G.; Milas, M.; Rinaudo, M.; Clarke-Sturman, A. J.Int. J.Biol. Macromol. 1990, 12, 195.

(6) Kido, S.; Nakanishi, T.; Norisuye, T.; Kaneda, I.; Yanaki, T.Biomacromolecules2001, 2, 952.

(7) Gravanis, G.; Milas, M.; Rinaudo, M.; Tinland, B.Carbohydr. Res.1987, 160, 259.

(8) Gravanis, G.; Milas, M.; Rinaudo, M.; Clarke-Sturman, A. J.Int. J.Biol. Macromol. 1990, 12, 201.

(9) Burova, T. V.; Golubeva, I. A.; Grinberg, N. V.; Mashkevich, A.Ya.; Grinberg, V. Ya.; Usov, A. I.; Navarini, L.; Cesaro, A.Biopolymers1996, 39, 517.

(10) Boutebba, A.; Milas, M.; Rinaudo, M.Biopolymers1997, 42, 811.(11) Borsali, R.; Rinaudo, M.; Noirez, L.Macromolecules1995, 28, 1085.(12) Boutebba, A.; Milas, M.; Rinaudo, M.Int. J. Biol. Macromol.1999,

24, 319.(13) Balnois, E.; Stoll, S.; Wilkinson. K. J.; Buffle, J.; Rinaudo, M.; Milas,

M. Macromolecules2000, 33, 7440.(14) Kaneda, I.; Kobayashi, A.; Miyazawa, K.: Yanaki, T.Polymer2002,

43, 1301.(15) Nakanishi, T.; Norisuye, T.Polym. Bull. 2001, 47, 47.(16) Kratky, O.; Porod, G.Recl. TraV. Chim. Pays-Bas1949, 68, 1106.(17) Yamakawa, H.; Fujii, M.Macromolecules1974, 7, 128.(18) Yamakawa, H.; Yoshizaki, T.Macromolecules1980, 13, 633.(19) Schmidt, M.Macromolecules, 1984, 17, 553.(20) Norisuye, T.; Fujita H.Polym. J. 1982, 14, 143.(21) Norisuye, T.; Tsuboi, A.; Teramoto, A.Polym. J. 1996, 28, 357.(22) Yamakawa, H.; Fujii, M.Macromolecules1973, 6, 407.(23) Odijk, T. J. Polym. Sci, Polym. Phys. Ed. 1977, 15, 477.(24) Skolnick, J.; Fixman, M.Macromolecules1977, 10, 944.(25) Le Bret, M.J. Chem. Phys.1982, 76, 6243.(26) Norisuye, T.Prog. Polym. Sci. 1993, 18, 543.(27) Tsuboi, A.; Yamasaki, M.; Norisuye, T.; Teramoto, A.Polym. J.

1995, 27, 1219 and references therein.(28) Kirkwood, J. G.; Goldberg, R. J.J. Chem. Phys.1950, 18, 54.(29) Stockmayer, W. H.J. Chem. Phys.1950, 18, 58.(30) See, for example, Sato, T.; Norisuye, T.; Fujita, H.J. Polym. Sci.,

Part B: Polym. Phys. 1987, 25, 1.(31) Yamakawa H.Modern Theory of Polymer Solutions; Harper &

Row: New York, 1971.(32) Yamakawa, H.Helical Wormlike Chains in Polymer Solutions;

Springer: Berlin, 1997.(33) Kawakami, K.: Norisuye, T.Macromolecules1991, 24, 4898.

BM020132F

Figure 10. Comparison between measured and calculated A2LS/A22

for sample S-4 in 0.01 M aqueous NaCl. Curve, eq A-4 with A11/A22

) 1.16 - 1.9f.

742 Biomacromolecules, Vol. 4, No. 3, 2003 Nakanishi and Norisuye