J. Biol. Macromol., 10 (1), 3-11 (2010)

Regular Paper
Differences in Excluded Volume, Diffusion Coefficient, and Surface Charge of Alginates withDifferent Mannuronate to Guluronate Ratio
Hideki Minami1, Jun’ichi Katakawa2, §, and Yoh Sano2
1

Hiroshima International University, 5-1-1, Hirokoshingai, Kure, Hiroshima 737-0112, Japanand 2Setsunan University, 45-1, Nagaotoge-cho, Hirakata, Osaka 573-0101, Japan

Received September 9, 2009; Accepted October 19, 2009
Abstract
The difference in excluded volume of two types of alginates having high mannuronate(M) content (500M) and high guluronate (G) content (500G) were measured with gel filtrationand viscosity together with dynamic and electrophoretic light scattering method.
molecular weight of both alginates was almost the same.

The

The pattern of gel filtration of 500M

eluted faster than 500G showing that the excluded volume of 500M is larger than 500G. The
reduced viscosity of 500M was larger than 500G.
smaller than 500G.

The diffusion coefficient of 500M was

Zeta-potential showing surface charge of molecular domain was about -60

mV for 500M and about -50 mV for 500G.

These results may be attributed to the fact that the

carboxyl groups of MM block of 500M exposed from the polymer chain are separated fromeach other and the 500M molecule takes the flexible structure as revealed by molecularmechanics program 3 (MM3).

On the other hand, the carboxyl groups of one guluronic acid of

GG block of 500G can form the hydrogen bond with the hydroxyl group on other guluronic acidand therefore the polymer chain of 500G takes more compact structure than the flexible
structure.

For these reasons, the excluded volume of 500G become smaller than 500M and thesurface charge of 500G molecule indicates smaller negative value than 500M.
Key words: alginate, mannuronate to guluronate ratio, excluded volume, diffusion coefficient,zeta-potential§To whom correspondences should be
addressed.
E-mail: katakawa@pharm.setsunan.ac.jp
Phone: +81-72-866-3150
Fax: +81-72-866-3150

Introduction
Alginate is an important commercial
polysaccharide prepared from brown algae. It
is a copolymer of 1,4-linked D-mannuronicSolution properties of two type alginates

4acid (M) and L-guluronic acid (G) consistingand high rich G content (500G) was

mainly of blocks of each and of alternating

discussed together with the data of diffusion

copolymer [1-4].

coefficient measured with dynamic light

The exact composition

depends on algal source, algal tissue and also

scatteringseason [1-4].

measured with electrophoretic light scattering

Alginate is used commerciallyfor its ability to form very viscous solutions

method,andsurfacechargemethod.

and gels with divalent and trivalent metal
cations.

Materials and Methods

The uronic acid residues in alginate are

Materials

arranged in the chains as homopolymericThesodiumalginates(bioreactor

blocks of M and G interspersed withgrade) were purchased from Kibun Food

sequences. The selectivity for cations and theChem. Ltd. (Tokyo, Japan) [7].

gel forming properties of alginate aremannuroic acid to guluronic acid residues

correlated with the content of G-blocks [4].

(M/G) was determined using the method of

The enzymes mannuronan C-5 epimerases

Huang et al. [2].

catalyse

Table 1.

thein-chainepimerizationof

The ratio of

The results are shown in

pH was measured with pH meter

β-D-mannuronic acid to α -L-guluronic(Mettler-Toledo type Seven Easy) at 1 mg/ml

acid in the last step of alginate biosynthesisalginate concentration in 10 mM NaCl.

[5].

The technologically important physicalproperties of alginate in solution and/or gel

Table 1 Analytical data of sodium alginate

are largely controlled by the relative amounts

samplesof the type of block present. Recently, Storz

Typeet el. [6]. analyzed about the ultra-high

Viscosity(mPa･s)

viscosity and highly biocompatible alginatepHin high salt buffer condition such as 150mM

M/G ratio

NaCl with fluorescence, NMR, viscometry-5

and multi-angle light scattering (MALS)
methods and showed that the monomer
composition of alginate had no effect on coil
expansion. However, the physical propertiesof alginate with different block compositions
M and G have not yet been studied in details.
In the present paper, the difference of the
excluded volume measured with gel filtrationand viscosity method of two types ofalginates having high rich M content (500M)

1)

2)

500M

500G

568

550

6.6

6.7

1.05

0.41

Mw×10
1.08
1.04
cited from catalog of Kibun Food Chem.
Ltd.(alginate concentration is 1 g/100 ml
at 20 ℃)2)
cited from catalog of Kibun Food Chem.
Ltd.
1)

The heavy metal was below 20 μg/g
in each sample. All other reagents were of thepurest grade available.

After two types ofalginate samples were dissolved in 0.01 M
imidazole aqueous solution (pH 7.0), sodiumMinami, Katakawa and Sanoions from alginates were removed with

Gel filtration

cation-exchange resin (Amberlite IR120B,
Organo, Japan) [8].

The concentration of5

Gel filtration was carried out on a
Sepharose 6B column (Amersham Pharmacia

alginate was determined by Phenol-sulfuric

Co. Ltd.) in 5 mM NaCl solution.

method and prepared with 1 mg/ml.

The

concentration in each elution tubes wascarboxylate content of this solution was

detected by optical density at 480 nm withdetermined by titration with NaOH and the

Phenol-sulfuric method [13].

Alginate

end point suggested one carboxylic acid per
one monosaccharide.

Viscosity
Viscosity measurements were carriedLight scattering measurements
Measurements of the intensity of theout with an Ostwald-type viscometer at 25 °C.
Flow time was about 120 s for pure water

light scattering were done with a modified[14].

The viscometer was at first rinsed

Ellipsometer, an automatic light scatteringwith concentrated nitric acid and then washedanalyzer AEP-100 (Shimadzu Co., Ltd.,exhaustively with pure water.

Japan), at 20°C [9-12]. The linearly polarized

solution of several concentrations was mademonochromatic incident light passed throughby diluting the stock solution with 10 mM

the cylindrical scattering cell, and the light

NaCl solution.

Each alginate

scattered at any scattering angle was detected
through the linear analyzer by a photo-

Dynamic light scattering measurement
The diffusion coefficients of alginate

multiplier in a telescope arm that can rotatefrom 45° to 135° by a stepping motor.
The sample was dissolved in 0.2 M

solutions were measured with the dynamic
lightscattering

instrument

of

a

light

NaCl aqueous solution and diluted using

scattering analyzer ELS-800 (Ohtsuka Co.,same buffer solution from stock solution toLtd., Japan) at 20 °C [11]. He-Ne laser ofdesired concentration of alginate and was

632.8 nm, and the scattering angle was 90°.

made optically clean by filtrating with a

The autocorrelation function of the photo-

Millipore filter (pore size, 0.45 μm). The

multiplier photocurrent was automaticallyapparatus constant was obtained by using aanalyzed

solution of bovine serum albumin in 0.1 M

techniques

phosphate buffer (pH 7.2), whose molecular

diffusion coefficient of each solution ofweight was taken as 65,800 optically clarified

alginate [14]. The sample was made opticallythrough a Gelman (Germany) filter (pore size,

clean by filtering with a Millipore (Germany)

0.2 μm).

filter (pore size, 0.45 μm).

bystandard

toobtaincomputerthefittingtranslational

Solution properties of two type alginates

6

Results and Discussion

Zeta-potential measurement
The zeta-potential of optically clean

Molecular weight of alginate samples

alginate by filtering with Millipore filter was
1.4

measured with laser electrophoretic zeta-

1.2

potential analyzer ELS-80 (Ohtsuka Co., Ltd.,
Japan) at 25 °C [15]. The photomultiplier1.0

was mounted on a computer-controlled

0.8

goniometer by which the scattering angle

0.6

could be varied over a range of 5-30°. An

0.4electrophoretic cell with a square cross

0.2

section was used. The dimensions cell were

0.0
0.0

17 mm in length (in the direction of applied
electric field), 10 mm in width (in that of theincident beam), and 2 mm in thickness (in

0.5
1.0
1.5
2.0
2.5
Concentration of alginate (mg/ml)

3.0

Fig. 1 Zimm plots of 500G (open circles)
and 500M (closed circles) in 0.2M NaCl bylaser light scattering methodthat vertical to both of them). The mountingof the electrophoretic cell was designed so

The molecular weight of both alginatethat it can be moved in the vertical direction

samples were determined by Zimm plot

and the scattering volume in the cell can bemethod of laser light scattering [16]. A

set at a mechanical precision of 0.01 mm.

grid-like Zimm plot method is normally used

The temperature of the cell was controlled bywith both the angular dependence correlated

the circulation of thermostated water throughtothe mounting block of the cell. The cellconcentration dependence associated withholder is provided with a stage, the height ofboth the molecular weight and the second

which is adjustable with an automatedvirial coefficient. The characterization of

micrometer so that the laser beam path can be

alginate samples such as in this paper is

moved from the top to the bottom of the cell.

especially a necessity in the case of the latter

Electroosmosis leading to flow with acase. The extrapolation of zero angle wasparabolic velocity profile cannot be avoided

done by the computer simulation method. As

in the case of a closed electrophoresis cell.

is shown in Fig.1, K’Ø2c/R0 was plotted

The true mobility can be calculated byagainst the weight concentration of alginate c,determining

atwhere K’ is the optical constant, Ø is thevarious positions and the data points wererefractive index increment and R0 is thefitted by the least-square method to obtain the

reduced scattering intensity at scattering

velocities at the stationary layer.

angle equal to zero. The Ø was used as 0.154

theapparent

mobilities

theradiusofgyrationandtheml/g for both alginate samples [17]. Fig. 1
gives the weight-average molecular weightMinami, Katakawa and Sano

from the intercept of the ordinate and the7

Gel filtration of alginate samples

second virial coefficient showing polymerpolymer and polymer-solvent interactions

0.8

from the inclination of the straight lines. The

0.7

data was little scattered. However, the0.6

concentration dependence of 500M was

0.5

appreciably little higher than 500G. The

0.4

linear extrapolation of zero concentration was
done with the computer simulation method.

0.3
0.2
0.1

The molecular weight of both alginate
samples was estimated as almost the sameand approximate 1.0 × 105, as is shown in
Table 1. The charged polyelectrolyte such as

0
0

50

100

150

200

250

300

Elution volume (ml)Fig. 2 Elution pattern of gel filtration of
500G (open circles) and 500M(closed circles)

alginate is normally good soluble in water.
Both alginate samples have a high negative

Fig. 2 shows the elution pattern of gel

charge and are highly soluble in water as afiltration

monomolecular molecule at the diluted

horizontal axis shows the elution volume andconcentration range. In this case, the second

the vertical axis indicates the optical density

virial coefficient shows a positive value dueof each fraction by phenol sulfuric acidto mainly the polymer-polymer repulsive

method. The 500M eluted faster than 500G.

interaction. At the much higher salt bufferThis means that the excluded volume of

condition, the alginate decreases the negative

500M is larger than 500G and the tertiary

surface charge due to the charge shielding

molecular domain structure of 500G takes

effect with salt. In this condition, the second

more compact structure than 500M, althoughvirial coefficient shows almost zero. If thethe molecular weight of both samples isalginate

almost same.

formsintermolecularly

the

of

both

samples,

where

the

association, the second virial coefficientshows normally a negative value [18]. In the

Viscosity of alginate samples

present paper, the second virial coefficient

The reduced viscosity, ηsp/c, wastook a positive value in both cases and was

calculated from the average values of three

attributed to mainly the negative charged

experimental times. Fig. 3 showed thepolymer-polymer interaction. The positive

concentration dependence of the reduced

second virial coefficient of 500M was larger

viscosity of both alginate samples. The

than 500G, because the negative surface

reduced viscosity of 500G decreased almost

charge of 500M molecule has much higher

straightly with the decrease of alginate

than 500G molecule as is discussed later.

concentration.

8

Solution properties of two type alginates

samples than the diluted measurements of gel
25

filtration chromatography and/or the reduced

20

viscosity in this paper.

15

Diffusion coefficient of alginate samples
The diffusion coefficient, D, of alginates10

was monitored with the quasi-elastic lightscattering method [11,14]. The experiments

5

0
0.0

were accumulated over 100 times for thesame samples and repeated 3 times. The data
0.5
1.0
1.5
2.0
2.5
Concentration of alginate (g/100ml) × 10-2

Fig. 3 Concentration dependence of reduced
viscosity of 500G (open circles) and 500M
(closed circles)
The reduced viscosity of 500M, however,increased monotonously with decreasing theconcentration of alginate because the buffer
was low ionic strength and 500M sample hashigher negative surface charge than 500G, as
is discussed later. The reduced viscosity of
500M was always larger than 500G. This
result is good consistent with gel filtration
chromatography, and the size of excluded
volume for 500M is larger than 500G.
Matsumoto and Mashiko [19] showedthat the values of the dynamic viscoelastic
moduli, the storage modulus and loss
modulus, are almost independent of M/Gratio of alginate and suggested that theviscoelastic properties of alginate aqueous
solution are strongly influenced by themolecular weight and not by M/G ratio ofalginate samples. This may be caused by thefact that the viscoelastic properties aremeasured at much higher concentration of thewere very reproducible.
The autocorrelater was connected to
the

light

scattering

autocorrelation

analyzerfunction

of

and

the

the

photo-

multiplier photocurrent was automaticallyanalyzed
techniques
diffusionbystandard

toobtaincoefficientcomputer
theoffittingtranslational

alginates.

Theobserved single-clipped photocount correlation function could be described by a single
exponential curve. The translational diffusion
coefficient was obtained by the first cumulantof the electric correlation function.
The
determined

diffusioncoefficientbyinitialautocorrelation

thefunction.

The

coefficient obtained was 2.51×10

was

slope

of

diffusion-10

cm2/secfor 500G and 1.76×10-10 cm2/sec for 500M,
respectively. The diffusion coefficient of
500G is larger than 500M, indicating that thesize of excluded volume of 500G is smaller
than 500M. This result is very goodcoincident with the results of gel filtrationand viscosity.

Minami, Katakawa and Sano9

because of having much G content. On the

Surface charge of alginate samples

other hand, the 500M alginate consists of0

many MM block because of having much M-10content. As is shown in Fig. 5, the carboxylgroups in MM block is exposed from the-20

polymer chain of alginate molecule and the-30

carboxyl groups exposed are separated from-40

each other because of the electrostatic-50

repulsive force, where the polymer chain of-60

alginate molecule takes the flexible structure

-70
0.000

[20]. On the other hand, the carboxyl group
0.005

0.010

0.015

0.020

0.025

0.030

NaCl/Alg(mol/mg)

Fig. 4 Zeta-potential of 500G (open circles)
and 500M (closed circles) by electrophoretic
light scattering method.

on one guluronic acid in GG block can form
the hydrogen bond to the hydroxyl group on
other guluronic acid, where the polymer
chain of alginate takes more compact
structure than the flexible structure and also

The study of electrophoretic behavior

the negative surface charge of alginateof alginates affords the important and uniquemolecule in GG block becomes smaller thaninformation about surface properties ofMM block.

alginate

molecule.

Zeta-potential

wasmeasured by electrophoretic light scattering
method. As is shown in Fig. 4, zeta-potential
was about -60 mV for 500M and about -50
mV for 500G, respectively. The negative
surface charge of 500M was much higher
than 500G. This result is coincident with thedata of the higher second virial coefficient
and

also

the

strongerconcentration

dependence of the reduced viscosity of 500M
than 500G.

Fig. 5 Schematic illustration of alginic acidby molecular mechanics program 3 (MM3).
Left chain shows GG block and right chain
indicates MM block.

Alginic acid is composed of GG block(guluronic acid-guluronic acid), GM block

For these reasons, it is concluded that(guluronic acid-mannuronic acid), MG block

the excluded volume of 500G becomes(mannuronic acid-guluronic acid), MM block

smaller than 500M and the surface charge of(mannuronic acid-mannuronic acid). The

500G molecule indicates smaller negative

500G alginate consists of many GG block

value than 500M.

Solution properties of two type alginates

10

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Communicated by Yoko Nitta