Nonperturbing Fluorescent Labeling of Polysaccharides

Biomacromolecules 2002, 3, 857-864

857

Nonperturbing Fluorescent Labeling of Polysaccharides Franc¸ oise Meunier and Kevin J. Wilkinson* CABE (Analytical and Biophysical Environmental Chemistry), Sciences II, 30 Quai Ernest Ansermet, 1211 Geneva 4, Switzerland Received February 18, 2002; Revised Manuscript Received March 26, 2002

A fluorescent labeling procedure, which does not perturb macromolecular conformations, was employed to bind a rhodamine derivative to the reducing end of several water-soluble polysaccharides by reductive amination in the presence of sodium cyanoborohydride. Fluorescence correlation spectroscopy, atomic force microscopy, and size exclusion chromatography were used to demonstrate that the conformations of the polysaccharides schizophyllan, polygalacturonic acid (PGUA), succinoglycan, and several dextrans were maintained following the labeling procedure. Introduction Novel fluorescence-based techniques such as fluorescence correlation spectroscopy (FCS) or scanning near optical microscopy (SNOM) are lowering the detection limits at which it is possible to determine important physicochemical parameters such as polymer conformations and sizes.1,2 It is therefore of great interest to fluorescently label polysaccharides without perturbing their structural conformation. Derivatization can be accomplished both by adsorption3-5 or by introducing covalent bonds between the fluorophore and the polysaccharide. While adsorption is least likely to perturb polysaccharide structure, covalent bonds are more likely to be stable, especially in the presence of changes in the ionic strength, temperature, or pH of the medium. On the other hand, covalent labeling generally requires potentially harsh conditions that might result in the degradation of the polysaccharide. Therefore, to avoid conformational modifications of the polysaccharide, reactions should be performed under minimally perturbing conditions of pH, temperature, and ionic strength. Furthermore, to ensure that molecular conformations are maintained whenever possible, labeling should be performed with only a small number of fluorophores for each macromolecule. In the past, several covalent labeling procedures have been described for polysaccharides. For polysaccharides containing carboxylic functional groups, the fluorescent label can be bound through an amide bond using a carbodiimide,6 but this functional group is not present in all polysaccharides. Amines can also be grafted to the hydroxyl functional groups of the polysaccharide7 using cyanogen bromide; however, the resulting polysaccharide chains are of variable fluorescence due to a varying fluorophore density along each macromolecule. In addition, the CNBr used for the derivatization is very toxic. Although it can be replaced by less toxic chemicals such as diisocyanate8 or ethylchloroformiate,9 the reactions need to be performed under more difficult anhydrous conditions. Finally, it is also possible to introduce an aldehyde on the polysaccharide using sodium periodate, 10

but this procedure can induce chain hydrolysis or alteration by rupture of monosaccharide rings. In addition, the procedure is not selective for fluorophore localization. In this study, derivatizations were performed on the reducing end groups of the polysaccharides in aqueous solution. The conformations of the polysaccharide/fluorophore conjugates were verified using FCS, tapping-mode atomic force microscopy (Tm-AFM), and size exclusion chromatography (SEC). Experimental Section Samples. Schizophyllan, kindly supplied by Dr. W. Itoh (Taito Co. LTD, Japan, Mw ) 437000 g mol-1),11 is a watersoluble and neutral extracellular polysaccharide. The main chain consists of a triple helix of β-(1f3)-D-glucans in which every third glucose residue in the main chain has one β-(1f6)-D-glucosyl side chain (Figure 1A). In water, the polymer adopts a triple helical structure, which can dissociate into single-stranded random coils at temperatures greater than 135 °C,12 NaOH concentrations exceeding 0.25 M,13 or DMSO concentrations exceeding 90% (v/v) with respect to water.14 Succinoglycan, kindly supplied by Dr. M. Milas (CERMAV, France), is an acid polysaccharide with a molar ratio of 7 D-glucose:1 D-galactose:1 pyruvate:0.75 succinate (Figure 1B). Finally, four dextrans (molar masses of 9900, 40000, 68400, and 464000 g mol-1) and a polygalacturonic acid were obtained from Sigma (Figure 1C-D). Native polysaccharides were dissolved in Milli-Q grade water (Millipore; R > 18 MΩ‚cm). Polysaccharide samples were stirred for 24 h prior to filtration through a 0.22 µm polyvinylidene fluoride membrane (Durapore, Millipore). Derivatization. Polysaccharides were derivitized using a modification of the method proposed by Jackson15 for the covalent tagging of the reducing end of oligosaccharides using 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS). Rhodamine green X-succinimidyl ester hydrochloride, R6113, from Molecular Probes (Figure 1E), was employed in this

10.1021/bm0255241 CCC: $22.00 © 2002 American Chemical Society Published on Web 05/07/2002

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Biomacromolecules, Vol. 3, No. 4, 2002

Meunier and Wilkinson

Figure 1. Structures of the repeating units of (A) schizophyllan, (B) succinoglycan, (C) dextran, (D) polygalacturonic acid, and (E) structure of the fluorophore rhodamine green X-succinimidyl ester hydrochloride (R6113).

Biomacromolecules, Vol. 3, No. 4, 2002 859

Fluorescent Labeling of Polysaccharides

Figure 2. Schematization of the derivatization by R6113, illustrated with the reducing end of schizophyllan.

study. This fluorophore was selected both for the amine functional group required for the labeling and for the excitation wavelength of 495 nm which allows its use with an argon ion laser. R6113 was dissolved in methanol to give a stock solution of 3.2 × 10-2 mol L-1. Excitation and emission spectra of the fluorophore and the labeled polysaccharide were obtained using a Jasco FP-750 spectrofluorimeter. One milliliter of a polysaccharide stock solution (2.3 × 10-6 mol L-1 for schizophyllan and succinoglycan and 2.3 × 10-4 mol L-1 for the others; molar concentrations were calculated using the weight average molar mass of each polysaccharide) was mixed with a variable volume of the R6113 stock solution and then adjusted to 2 mL with a mixture of acetic acid/water (15/85 v/v) in a screw-capped polypropylene tube to obtain a solution pH of 2.3. In this manner, the fluorophore/polysaccharide molar ratio was varied from 10 to 1000 for schizophyllan and was fixed at 500 for the other polysaccharides. The mixture was subsequently heated for 30 min at 80 °C. Twenty-five microliters of freshly prepared 9.2 × 10-2 M sodium cyanoborohydride solution (Aldrich) dissolved in dimethyl sulfoxide (DMSO) was then added to the mixture to give a 1000-fold excess of NaBH3CN with respect to the polysaccharide. The tube was incubated for 90 min at 80 °C on a rotary shaker. The reaction scheme between the amine functional group of the fluorophore and the aldehyde functional group of the polysaccharide, summarized in Figure 2, consisted of the formation of an imine followed by reduction to an amide. After cooling, the labeled schizophyllan and dextran were purified from nonreacted R6113 by three extractions with an ethanol/water solution (75/25 v/v) followed by a 30 min centrifugation at 3000g. The final product was dissolved in a small volume of water so that the final solution of labeled polysaccharide was concentrated 2-fold. Since PGUA and succinoglycan are polyelectrolytes, they precipited only upon addition of NaCl. Excess free fluorophore was removed using dialysis (nominal cutoff 3500 g mol-1). Fluorescence Correlation Spectroscopy (FCS). FCS is based on a time-resolved analysis of fluorescence intensity fluctuations. The system is theoretically sensitive enough to allow single molecule detection. The apparatus employed here, a Confocor (Carl Zeiss Jena), consisted of an inverted microscope, an Axiovert 135 TV, with a C-Apochromat 40×, N.A. 1.2 objective lens, an argon ion laser (LGK 7812 ML), an avalanche photodiode (SPCM-200-PQ, EG&G) in the single photon counting mode, and a digital correlator (ALV 5000/E, ALV GmbH).

Analysis was realized at an excitation wavelength of 488 nm using an Ar ion laser. Fluctuations in the measured fluorescence intensity are attributed to the translational diffusion of the fluorescent molecules in and out of a small confocal volume (average 0.8 µm3) formed by the highly focused laser light and defined by the distances from the center to the edge of the confocal volume in the radial, ω1, and axial, ω2, directions. Variations in the fluorescence intensity are quantified using one of two autocorrelation functions:16 For a single component:

( )(

G(t) ) G(0) 1 +

t τ1

-1

1+

)

t p τ1 2

-1/2

(1)

For two components: G(t) )

( ( )) ( ( ))

1 1 1-y t N t 1 + 1 + p2 τ1 τ1

1/2

+

y

1+

1

1/2

t t 1 + p2 τ2 τ2

(2)

where G(t) is the autocorrelation function, G(0) the autocorrelation function at time 0, t the delay time, τ1 the diffusion time required for R6113 to pass through the volume element, τ2 the diffusion time for the fluorescent polysaccharide, y the proportion of fluorophores that are attached to the polysaccharide, p the structural parameter, p ) ω2/ω1, and N the number of particles present in the confocal volume. The structural parameter was calibrated prior to each experiment using rhodamine 6G which has a known diffusion coefficient, D, of 2.8 × 10-6 cm2 s-1.17 For each sample, 10 runs of 100 s each were averaged. Means and standard deviations are reported throughout the paper. Fluorophore or polysaccharide-bound fluorophore concentrations were adjusted between 2.3 × 10-8 and 9.2 × 10-6 mol L-1 in order to avoid photodiode saturation. The diffusion coefficient of the R6113 fluorophore was determined in the absence of polysaccharide (eq 1) for use in the two-component model (eq 2). The diffusion coefficient of the polysaccharides was determined from the measured diffusion times, τ2 (eq 3). The equivalent hydrodynamic radii, rH, were estimated using the Stokes-Einstein equation (eq).18 For polysaccharides occurring as rigid rods (e.g., schizophyllan), the estimated total length (L) can be calculated directly using the Broersma equation (eq 5)19,20 ω12 4τ2

(3)

kT 6πηD2

(4)

kT A 3πηD2

(5)

D2 ) rH ) L)

where k is the Boltzmann constant, T is the absolute temperature, D is the diffusion coefficient, and η is the viscosity of the solution (g cm-1 s-1). The subscripts 1 and 2 refer to the fluorophore and the labeled polysaccharide, respectively. The value of the constant, A, was determined

860



Table 1. Macromolecular Lengths and Widths Employed To Calculate Axial Ratiosa polysaccharide

length (nm)

diameter (nm)

P

A

Lp (nm)

triple helix

21027

1.0828

single chain dimer single chain

1.738 0.3223 0.6423 0.3

194.4 123.5 847 423 420

4.97 4.06 6.61 5.83 5.52

16127

21027 271 271 126

conformation

schizophyllan succinoglycan PGUA

1923 10523 936

a Values were extracted from the literature or estimated from molecular modeling using known molar masses. The constant A was determined using eq 6.

using length/diameter, a calculated axial ratio P, of the polysaccharide (eq 6) A ) ln(2P) + 0.017 +

5.8 9.154 2 ln(2P) ln(2P)

(6)

Values for the axial ratios were determined from published lengths and widths and from calculations using the molar masses (in Table 1). PGUA and succinoglycan have carboxylic groups in their chains, so under some conditions of pH and ionic strength, they were able to adopt semirigid conformations. Using the assumption of a wormlike chain, contour lengths can be estimated using Hearst’s relationship (eqs 6 and 7).21,22 For large persistence lengths (rigid rods) D)

L a kT ln + 0.166λL - 1 + 3πη0L a d

( ()

)

(7)

and for small persistence lengths (flexible coils) D)

Scientific). The polysaccharide end-to-end lengths, Lee, and contour lengths, L, were determined directly using the software. The number average contour lengths, Ln, and weight average contour lengths, Lw, were determined according to eqs 9 and 10

∑i niLi/∑i ni

(9)

∑i niLi2/∑i niLi

(10)

Ln )

a kT 1.843(λL)0.5 - ln(λa) - 2.431 3πη0L d

(

Figure 3. Fluorescence spectra for 10-8 M (s) fluorophore R6113 and for 10-7 M (‚ ‚ ‚) schizophyllan (partially labeled).

)

(8)

where λ is ) 1/2LP, a is the monomer length, and d is the diameter of the monomer. Size Exclusion Chromatography (SEC). Size exclusion chromatography was performed using an OH-pak SB-805 HQ analytical column and an OH-pak SB-LG pre-filter. Products were eluted at a constant flow rate of 0.5 mL min-1 using a Jasco PU-980 pump and evaluated successively with refractive index (RI, Jasco RI-930) and fluorescence (MerckHitachi F-1050) detection. Excitation and emission lengths were fixed at 490 and 530 nm, respectively, using windows of 15 nm. The eluent used for the analysis was 0.1 M NaNO3 that was filtered (0.22 µm) and sonicated (15 min). Due to the use of the RI and fluorescence detectors in series, peaks were corrected with respect to the dead volumes of the detectors. Tapping-Mode Atomic Force Microscopy. Five microliters of a 4.6 × 10-9 mol L-1 solution of schizophyllan or succinoglycan was dissolved in water and then deposited on 1 cm2 of freshly cleaved mica. The solutions were dried for 30 min under ambient conditions in an enclosed Petri dish. Samples were analyzed using a Nanoscope III multimode microscope from Digital Instruments (Santa Barbara, CA) using previously described conditions.23 The section analysis software of the microscope was used to determine the height of the polysaccharides. AFM images were digitalized using Scion image analysis software (Vbeta3b, based on NIH image software, Scion Corp.) and were analyzed with Sigma Scan Pro image analysis (Jandel

Lw )

where ni was the number of each chain. Polysaccharide persistence lengths, Lp, were deduced from the change in tangent direction of each vector link, θ, along the polysaccharide macromolecular contour distance23,24 〈θ2〉 ) l/Lp

(11)

where l represents the length of each vector link and 〈θ2〉 represents the mean square angular dependence. Due to the resolution limits of the digitalized AFM images, the polysaccharide lengths were determined with an error of (2 nm for the number average contour lengths and (6 nm for the weight average contour lengths. In any case, these errors are greatly overwhelmed by the sample polydispersity. Standard deviations are reported for all polymer measurements. Results and Discussion In the following section, the derivatization technique is developed in detail for schizophyllan and then confirmed with more targeted analyses on the other polysaccharides. Determination of the Excitation and Emission Wavelengths of the Conjugate. R6113 exhibited maximum excitation and emission wavelengths at 495 and 524 nm, respectively (Figure 3). The binding of the fluorophore to schizophyllan did not significantly modify these values.

Fluorescent Labeling of Polysaccharides


Figure 4. Chromatograms of native schizophyllan (s) and schizophyllan extracted with an ethanol/water mixture then redissolved in water (‚ ‚ ‚). Data were obtained using refractive index (RI) detection with a 0.1 M NaNO3 eluant.

Figure 5. Chromatographic profiles of schizophyllan derivatized with the fluorophore R6113 using refractive index (A) and fluorescence (B) detectors. The eluant was 0.1 M NaNO3.

Size Exclusion Chromatography. Assuming no adsorption of the polysaccharide to the column, size exclusion chromatography provides information on the hydrodynamic radii of the macromolecules.25 Because the unbound R6113 was diluted following purification and had a tendency to be adsorbed to the column, it was impossible to precisely quantify using this technique. On the other hand, the peaks provided by RI detection and corresponding to schizophyllan did not appear to be modified after purification by successive precipitation in ethanol (Figure 4). The presence of two shoulders on the relatively wide peak likely reflected sample polydispersity. Chromatograms using RI detection were also similar following derivatization (Figure 5) suggesting that the polysaccharide was not altered by the labeling process. A second chromatogram, obtained using a fluorescence detector (Figure 5) was observed to have a similar profile to that detected using RI detection. The similarity of elution profiles suggested that the fluorophore was bound to all size fractions of the polysaccharide. Similar behavior was observed for the other polysaccharides (Figure 6). Derivatization with the fluorophore R6113 did not change the elution times obtained by RI, and similar chromatograms were obtained by RI and fluorescence detection for the labeled polysaccharides. Schizophyllan is a long-chain polysaccharide containing one reducing end that is only accessible in the open conformation of the molecule. Because only a small proportion of the polysaccharide is in the open conformation at any given time, i.e., below 0.05%,26 it was important to introduce a large excess of fluorophore to ensure the efficiency of the derivatization. Experiments were performed using molar ratios of fluorophore which varied from 1 to 1000. Optimal labeling was obtained for a fluorophore/

Figure 6. Chromatographic profiles of dextran (Mw 9900), polygalacturonic acid (Mw ∼74000), and succinoglycan (Mw ∼350000) before and after their derivatization with the fluorophore R6113. The eluant is 0.1 M NaNO3. Detection was performed with RI prior to derivatization (A) and with RI (B) and fluorescence detection (C) following derivatization.

Figure 7. Evolution of the fluorescence intensity of the schizophyllan/ R6113 conjugate as a function of increasing R6113. The concentration of schizophyllan was 1.1 × 10-6 mol L-1.

schizophyllan ratio exceeding 300 (Figure 7). The fluorescence intensity of a 10-7 M solution of “labeled” schizophyllan was comparable to the intensity of a 10-8 M solution of fluorophore alone (Figure 3). Since only one fluorophore can be attached to each polysaccharide chain, this indicated that a maximum of 10% of the schizophyllan chains were derivatized under these conditions. Tapping Mode Atomic Force Microscopy (Tm-AFM). Tm-AFM images of native schizophyllan and labeled schizo-

862



Figure 8. TM-AFM images of (A) schizophyllan alone, (B) schizophyllan labeled with R6113, (C) succinoglycan alone, and (D) succinoglycan labeled with R6113. For schizophyllan, the scan size is 3 µm × 3 µm and for succinoglycan 1 µm × 1 µm.

phyllan dissolved in water are shown in panels A and B of Figure 8. Under the conditions of observation for the AFM, it is likely that the polysaccharides retained their inner shell of hydration water and thus maintained their conformations, although some modification might have been possible due to the interaction with the mica substrate. The AFM images confirmed that neither significant aggregation nor hydrolysis was induced by the labeling procedure. Since only one fluorophore was bound to each chain, it is unlikely that observation was biased by varying affinities of the labeled or unlabeled polysaccharides, i.e., the affinity of the tagged polysaccharide for the mica is unlikely to be any different than that for native schizophyllan. Persistence lengths, determined from several images, averaged 142 ( 20 nm for the conjugates and 149 ( 21 nm for schizophyllan alone. These values were not significantly different (Students t test, P < 0.05) which suggested that the polysaccharide was not degraded even following 2 h at 80 °C. This is consistent with the observations of Yanaki et al.12 who suggested that the triple helical rod was stable up to 135 °C. Furthermore, number average contour lengths, weight average contour lengths, and end-to-end distances of

the polysaccharide were unchanged after derivatization (Table 2) and were consistent with the values previously determined by Stokke.27 Finally, the chain thickness (AFM heights) varied from 0.80 to 0.88 nm for isolated chains to between 1 and 1.4 nm when small aggregates were observed. These values were comparable to the diameters of 1.08 nm observed by McIntire et al.28 for schizophyllan by a similar technique. For the most part, measured polysaccharide dimensions corresponded well to published values, with small observed differences being most likely due to sampling differences among the different techniques and research groups. Native and labeled succinoglycan were also observed by this technique (panels C and D of Figure 8). Their number average contour lengths were respectively 110 ( 35 and 111 ( 41 nm (Table 2), which also suggested that the polysaccharide was unchanged after derivatization. Furthermore, weight average contour lengths and end-to-end lengths were comparable, supporting the observation that the molecules were not influenced significantly by the reaction conditions. Fluorescence Correlation Spectroscopy. Fluorescence correlation spectroscopy allows the detection of fluorescent


Fluorescent Labeling of Polysaccharides Table 2. Analysis of Tm-AFM Imagesa native polysaccharide

labeled polysaccharide

lit. values

Schizophyllan no. av contour length, nm wt av contour length, nm end-to-end lengths, nm persistence lengths, nm heights/molecular diameter, nm

155 ( 89 207 135 ( 69 149 ( 21 0.80 ( 0.23

no. av contour lengths, nm wt av contour lengths, nm end-to-end lengths, nm

110 ( 35 121 109 ( 34

125 ( 76 180 124 ( 62 142 ( 20 0.88 ( 0.17

16427 21027

111 ( 41 126 115 ( 53

radius of gyration estimated at 63.9 nm38 for Mw ) 350000.

161 ( 3027 1.0828

Succinoglycan

a

Comparison of schizophyllan and succinoglycan before and after derivatization. Means and standard deviations are reported.

Table 3. Determination of Diffusion Coefficients and Hydrodynamic Radii by Fluorescence Correlation Spectroscopy for Schizophyllan, Succinoglycan, Polygalacturonic Acid, and Several Dextrans Labeled with the Fluorophore R6113 and Dissolved in Watera polysaccharide

Mw

conformation

D (10-12 m2 s-1)

rH (nm)

L1 (nm)

L2 (nm)

7.6 ( 0.4

28.0 ( 1.4

329 ( 16 284 ( 13 273 ( 30

418

245 ( 28 91 ( 4

137 302

schizophyllan

437000

rigid chain

succinoglycan

350000

mixture of semiflexible chains and rigid dimers

19.4 ( 1.7

11.1 ( 1.1

PGUA dextran

74000 464000 68400 40000 9900

coil or semirigid chain coil coil coil coil

29.2 ( 3.3 21.8 ( 1.7 31.5 ( 1.5 43.5 ( 2.9 67.0 ( 5.2

7.3 ( 0.8 9.9 ( 1.3 6.8 ( 0.4 5 ( 0.3 3.2 ( 0.3

600

a r values correspond to the equivalent hydrodynamic radii under the assumption of a compact sphere calculated using the Stokes-Einstein equation. H Contour lengths, L, were determined using the Broersma19,20 equations for rigid rods, L1, or the Hearst equations21,22 for a wormlike chain, L2. Due to the assumptions required by each of the models, not all of the calculations are appropriate in all cases. Nonetheless they are given for comparatives purposes only.

molecules with a sensitivity of about 10-9 M. Furthermore, the technique permits the differentiation of two components when their diffusion times differ by a factor of at least 1.6.29 For all samples, only a small amount of free fluorophore remained after purification by ethanol precipitation. Several measurements showed that the concentration of schizophyllan, which varied from 2.3 × 10-8 to 1.1 × 10-6 mol L-1, had no influence on the measured diffusion times. Furthermore, for a fluorophore/schizophyllan ratio which varied from 10 to 1000, neither diffusion coefficients nor the proportion of fluorophore bound to the schizophyllan was modified. The average value of the diffusion coefficient for schizophyllan was determined to be (7.6 ( 0.4) × 10-8 cm2 s-1. This result is in good agreement with the empirical prediction of Zentz,30 who estimated the diffusion coefficient of several molar masses of schizophyllan using the formula: D ) (1.86 × 10-3)Mw(-0.78) (D expressed in cm2 s-1). Using this formula and the known molar mass, Mw, of our sample, 437000 g mol-1,11 gave a value of 7.4 × 10-8 cm2 s-1, within experimental error. Average values for the contour length for schizophyllan were estimated to be between 284 and 329 nm using FCS. These values were obtained from the ranges of axial ratios determined from literature values (Table 1). These lengths were significantly larger than values published previously using transmission electron microscopy31 or from our AFM observations. This result was nonetheless anticipated since the FCS technique measures all of the molecules in solution while the microscopic techniques often eliminate molecular aggregates from the image analysis. Indeed, contour lengths

Figure 9. Diffusion coefficients of several polysaccharides as a function of their molar masses: (×) literature values of pullulan;33 and labeled polysaccharides analyzed by fluorescence correlation spectroscopy: ([) dextran/R6113, (0) PGUA/R6113, (4) schizophyllan/R6113, (b) succinoglycan/R6113.

between 162 and 188 nm were estimated from FCS for the schizophyllan/R6113 conjugate dissolved in 0.1 M borate (pH 9.3). These values were significantly smaller than lengths determined in water and more in line with literature values (Table 1). Borate is known to adsorb to polysaccharide cisdiols,32 which would produce negatively charged chains and dissociate small aggregates. Diffusion coefficients obtained by FCS for polygalacturonic acid, succinoglycan, and several dextrans were plotted as a function of molar mass (Figure 9). Diffusion coefficients decreased with the molar mass of the polysaccharide but not irrespective of their conformations. Diffusion coefficients of dextran, which adopts a coil conformation, varied in a similar manner as pullulan33 (Figure 9). Both the diffusion coef-

864



(mainly hydrodynamic) measurements of the polysaccharide conformations were not modified by the derivatization procedure. Acknowledgment. This work was supported by the Swiss National Fonds (project numbers 2000 - 050629.971, and 2100 - 055 668.981). We thank J. Buffle, D. Mavrocordatos, K. Startchev, and S. Stoll for helpful discussion. References and Notes Figure 10. Comparison of the hydrodynamic radii of several dextrans (], ref 34; ×, ref 35; 2, measured values for dextran/R6113 conjugate).

ficients and hydrodynamic radii of the dextran/R6113 conjuguate were equivalent to previously published values of the unmarked macromolecules34,35 (Figure 10). On the other hand, for similar molecular weights of about 450000 (Table 3), diffusion coefficients for dextran were higher than diffusion coefficients of schizophyllan, in reasonable agreement with their molecular compactness. Indeed, despite their similar molar masses, schizophyllan is much more elongated due to its capacity to form a triple helical rod under the conditions examined here. For similar molar masses of 70000, the equivalent hydrodynamic radius of PGUA was 7.3 nm, approximately the same as the dextran at 6.8 nm. Since, PGUA is a weak polyacid in water, it was probably partially extended at these pH values (ca. pH 6). As would be expected for a polyelectrolyte of this nature, the hydrodynamic radius of PGUA was observed to decrease at lower pH values, giving 5.3 ( 0.3 nm at pH 4. In water, succinoglycan has been previously described as a mixture of semiflexible and semirigid chains21 with coexisting single chains, dimers, and aggregates. The total length calculated from the diffusion coefficient, using eq 5, was 273 nm (Table 3). Once again, the FCS-derived value was significantly greater than the measured AFM length, probably due to the unavoidable presence of small aggregates in solution. Where appropriate, the contour lengths of the polysaccharides were also evaluated with Hearst’s equations21 (Table 3). Contour lengths largely exceeded expected values. For both schizophyllan and succinoglycan, contour lengths were 1.5- to 2-fold greater than literature values reported in Table 1.

(1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) (26) (27) (28) (29) (30) (31) (32)

Conclusion

(33)

The rhodamine derivative, R6113, was covalently bound to the reducing end groups of several polysaccharides. Classical fluorescence showed that the presence of fluorophore did not modify the fluorescence of R6113. Analysis using SEC, FCS, and Tm-AFM indicated that the molecular

(34) (35) (36) (37) (38)

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BM0255241

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