SOME PHYSICOCHEMICAL PROPERTIES OF SPECIFIC
           POLYSACCHARIDES*

BY MICHAEL HEIDELBERGER AND FORREST E. KENDALL
(From fhe Department of Medicine, Presbyterian Hospital and College oj
       Physicians and Surgeons, New York)

      (Received for publication, March 27, 1931)
In recent years the study of immunologically specific poly-
saccharides has not only contributed to the understanding of
bacterial specificity (1) but has also proved of purely chemical
interest because of the unusual structure of certain members of
the group. Thus the specific polysaccharide of Type III pneumo-
coccus has been shown to be a polyaldobionic acid built up of
glycurono-glucose units (2, 3).  It is, moreover, a typical colloid
in the sense that it does not diffuse through collodion or parchment
into water, and is, by virtue of its structure, so strong an acid that
its aqueous solutions turn Congo red paper blue.  Hence it seemed
of interest to study some of the physicochemical properties of solu-
tions of its salts, the more so as little is known of the behavior to
be expected of an organic, colloidal, strong electrolyte devoid of
basic groups. Accordingly, the sodium salt of the Type III pneu-
mococcus specific polysaccharide was subjected to a study of its
viscosity, conductance, and its behavior on diffusion. Measure-
ments were also made on solutions of other specific polysaccharides.
The diffusion data are reserved for a forthcoming paper dealing
with the molecular weight of specific polysaccharides, in which
connection the data recorded in the present communication will
again be taken up.
Physicochemical studies have previously been made of gum
arabic, a carbohydrate of a character somewhat analogous to that
of the Type III pneumococcus polysaccharide. This substance,
however, is the salt of a much weaker acid than the Type III
* The work described in this communication was carried out under the
Harkness Research Fund of the Presbyterian Hospital, New York.
                    127


128      Specific Polysaccharides

pneumococcus carbohydrate, since the equivalent weight of arabic
acid is about 1200, while the Type III substance (hereinafter
referred to as S III) possesses one carboxyl group for every 340 of
molecular weight.  Thomas and Murray (4) have given a titration
curve of arabic acid and also studied its osmotic pressure and vis-
cosity at various hydrogen ion concentrations, and concluded that
its behavior might be satisfactorily explained on the basis of the

98

96

94

92

90

08

86

04

82

80

70

7b

74

72

Fxa. 1. Conductance of sodium salt of pneumococcus Type III specific

polyseccharide.

Donnan equilibrium.  The application of this conclusion to the
viscosity data was rejected by Taft and Malm (5) on the basis of an
extended study which included conductivity measurements.
These resulted in the conclusion that the current was carried
mainly by the inorganic ions present.


m.
0       0
1.0631    3.3362
10.6849   39.059
20.2357   73.972
35.9751   131.507
66.4993.  243.088
105.4481   385.466

v"`% (at

ml.    epuiwienls           oh?u
929.7   0       0     9917.0
930.7   0.041755   0.647   9321.5
949.4   0.41534   2.04    6236.6
949.9  0.77873   2.79    4859.0
965.4   1.3822    3.69    3597.8
995.6   2.4416    4.94    2460.8
1034.2   3.7272    6.11    1895.0

TABLE I

Condwrtitity of Na Salt of S III at W f OAW~~

c x 101   loo fi

101
F

1.00337  0.01018
1.07279  0.07460
1.59969  0.59256
2.05804  1.0599
2.77948  1.7813
4.06372  3.0655
5.54017  4.5429

mh
0.005996
0.043936
0.34895
0.62420
1.0491
1.8054
2.6750

equivalents

Concentration of stock solution, 3.6555 X 10m6 p;m solution in air.
Equivalent weight, 349.
The cell was shunted by a resistance coil (S) of 16018.1 ohms in the measurements.
R. bridge readina.  R'. actual resistance of cell.  K, cell constant = 0.58395.

1' 1- 1  - K'
      - L'                   1009L
$=fi--$  v-;L=L'-LHG;A=-.
                            c

Specific con-
ductance COT-
mad for wee
(L) x 101

mhm

0.037940
0.34295
0.61820
1.0431
1.7994
2.6690

1
rc









-

Ekpivalent
onductnncc
(A)

90.863
82.571
79.385
76.575
73.697
71.609


130     Specific Polysaccharides

EXPERIMENTAL

Conductivity Determinations-The conductivity data were ob-
tained with the arrangement of apparatus described by Shedlovsky
(6).  An S III solution was added to a new type of cell (7) from a

                   TABLE 11
Viscosities of S III Measured with Bingham Viscometer, No. ids,
             K - 1.99 x lo-6

f  4
f *
8    z      solvent  i   Time 1    a    lr
6 c!                                    7-v   w
4        P Ii 7
                                 8
                      a              P
--             -------

`C.  per cm!             b-m. Pm  WC.   csn'i-  centi-
                    nq. cm.       poiasr  pi$SS
20 2.93   HIO      188.906859 25.78 1.005025.65 24.65 8.41
   0.293    `(       117.39 1390.6 3.248 1.0050 3.232 2.232 7.62
   0.293    `(      216.4 752.4 3.2341.0050 3.218 2.218 7.57
   0.0293   "      214.05 352.7 1.5031.0050 1.495 0.495 16.89
   0.0293   "       99.18 799.7 1.5391.0050 1.531 0.53118.12
   0.0293   "      280.3 266.1 1.4841.0050 1.477 0.47716.28
30 0.0293   "       99.12 617.9 1.2190.8007 1.522 0.522 17.82
   0.0293   "      208.99 286.4 1.1910.8007 1.488 0.48816.66
   0.0293   "      280.32 211.3 1.1790.8007 1.472 0.472 16.11
20 0.0293  0.17 M NaCl  280.04 192.8 1.0741.0090 1.064 0.064 2.18
   0.0293  0.17 "   "   193.23 278.7 1.0721.0090 1.082 0.062 2.12
   0.0293  0.17 "   "   110.87 487.0 1.0751.0090 1.065 0.065 2.22
30 0.0293  0.17 "   I1   111.08 388.3 0.8580.809 1.061 0.061 2.08
   0.0293  0.17 "   "  212.37 202.8 0.8570.809 1.059 0.059 2.01
20 0.0586  0.17 `I   "  233.86 243.7 1.1341.009 1.124 0.124 2.12
   0.0586  0.17 "   "   132.31 431.4 1.1341.009 1.124 0.124 2.12
   0.0293  0.85 "   "  132.25 426.9 1.1241.078 1.042 0.042 1.43
   0.0293  0.85 "   "  166.79 338.4 1.123,1.078 1.042 0.042 1.43
   0.0293  0.85 "   "  219.27 257.8 1.124/1.078 1.043 0.043 1.47

weight burette, so that increasing concentrations were measured
without emptying the cell. The equivalent weight was taken as
340 and measurements were made in the range 0.039 to 3.85 milli-
equivalents. The extrapolation to limiting conductance was made
with the aid of a derived slope curve by use of the formula given
in Fig. 1.  The data are recorded in Table I and Fig. 1.
That the results were not influenced by hydrolysis is indicated by


    M. Heidelberger and F. E. Kendall    131

the fact that no shift in pH could be observed with either methy
red or pheno1 red as indicator on diluting a 30 mM solution 100
times with freshly boiled distilled water.

TABLE III

Viscosities oj S III Solutions.  Ostwald Viscometer at ZOO"

Subetgnce

Ha0
s III

KC1

s III

KC1
s III

NapHPO,
s III

HCl
s III
HCI
s III
N&l
s III

sdvmt

HIO
"
"
1`
"
1`

60

1.21 Y KC1
1.21 " "
Ha0
3.35 Y KC1
3.35 " `<
Ha0
0.05 M NalHPO
0.05 " "
Hz0
0.27 Y HCl
HIO
2.60 M HCI
Ha0
0.17 M NaCl

* Corrected for relative de] nei ty of solution, 4", - 1.003; Tab.. X d.

------
per cent .adc. MC.
     92.6
0.293 303*  92.63.272.27 7.75
0.0580 174.8 92.6 1.890.89 15.20
0.0293 147.9 92.6 1.600.6020.48
0.0195 137.1 92.61.480.4824.62
0.01465 132.5 92.6 1.430.4329.35
0.01172124.4 92.61.340.3429.10

1.21$36.6~ / 1 j

0.2930 129.9 86.61.500.50  1.71 1
0.0293 90.5 86.61.050.05 1.71
3.35 Y 82.4
0.293 99.4 82.41.210.21 0.72
0.0293 84.3 82.41.020.02 0.68
0.05 M 95.6
1.00 406.7 95.54.263.26 3.26
0.10   117.0 95.51.230.23 2.30
0.27 M 94.1
0.0293 97.7 94.11.040.04
2.60 M 101.5
0.0293 106.1101.5 1.050.05
0.17 M 93.7
0.0293 99.6 93.7 1.060.06

b
1









-

4.19
3.68
3.50
3.44
3.55
3.14

Q-P
7
orrectad
`or vol-
ume of
KC1

1.65

0.64

The conductivity determinations were made at The Rockefeller
Institute for Medical Research by Dr. T. Shedlovsky, and it is a
pleasure for the writers to express their indebtedness to him for his
freely given time and invaluable aid in the interpretation of the


132      Specific Polysaccharides

data.  Thanks are also due Dr. Duncan A. MacInnes of the Rocke-
feller Institute for his interest and assistance.

TABLE IV

Viscosities of S I and S II in Ostwald Viscometer at 80'

Sub-
Lance

Solvent

SO
SI

Hz0
"
`I
"
"
"

KC1   H20

SI

1.34 M KC1
3.35 " "
0.17 " NaCl
0 27 " HCL
2:m ,` "
0.008 M HCl
0.009 " "

Hz0
s II

NaCl
s II

Hz0
`I
`t
0.85 M NaCl

-



. -









I
-

concen- Time
t&ion

--
parcsnt 1)6c.
     92.9
0.3273 187.9'
0.06546 122.3
0.03273 108.7
0.02181105.6
0.01637 103.2
0.0131 109.5

92.9  2.05  1.05  3.21  1.84
92.9  1.32  0.32  4.89  1.25
92.9  1.17  0.17  5.20  0.94
92.9  1.14  0.14  6.42  0.96
92.9  1.11  0.11  6.72  0.86
92.9  1.08  0.08  6.11  0.70

1.34 116 86.0

0.3273 116.5  86.0
0.3273 109.6  82.4
0.3273 132.8  93.7
0.3273 141.4  94.1
0.3273 153.0  01.5
0.3273 164.5  92.6
0.2975 146.2  92.6

     93.1
1.106 113.71
0.221 97.2
0.85 M 96.5
0.553 103.2

93.1
93.1

96.5

-



--

?P
c

1.35  0.35  1.07
1.33  0.33  1 .Ol
1.42  0.42  1.28
1.50  0.50  1.53
1.51  0.51  1.56
1.78  0.78  2.38
1.58  0.58  1.95

1.22
1.04


1.07

0.22
0.04


0.07

0.20
0.18

,
I


I
-

0.13

"sp
de

`)SP
c
orlmti
`or vol-
umeof
KC1

1.03
0.90

o Corrected for relative density of solution, 4 = 1.002.
t From Table III.  Time for Ha0 is used ??? 0.908 and 0.009 M HCl.
$ Corrected for reIative density of solution, d: = 1.097.

Viscosity Measurements-The viscosity data given in Table II
were determined as described by Bingham (8) with the Bingham
viscometer at Lafayette College, and the writers wish to thank
Professor E. C. Bingham for extending to them the hospitality of
his laboratory and the benefit of his personal interest.  All pres-


M. Heidelberger and I?. E. Kendall    133

sures were corrected for temperature, hydrostatic head, and flow.
The viscosities were expressed in absolute units and the relative
and specific viscosities calculated.

The remaining viscosities were determined with an Ostwald
viscometer at 20", the value obtained with an S III solution within
the range of the Hagen-Poiseuille law checking closely with that
found with the aid of the Bingham instrument.  5.0 cc. of solution
were used in every case.  Potassium chloride was used instead of
sodium chloride at the higher salt concentrations since its effect on
the viscosity of water is almost negligible.

TABLE V

V'iscosities of Specific Polyaaccharides in Ostwald Viscometer at 80"

Specific gum arabic (S.G.A.)

       per cent    8CC.                UC.
&O          93.1
S.G.A.   4.85    143.2  1.022  146.4  93.1   1.57   0.57   0.12
"    0.485    99.0  1.002   99.2  93.1   1.07   0.07   0.14
  I`    0.0435   94.0            93.1   1.01   0.01   0.21

  Bovine tubercle bacillus polysaccharide (B.B.G. 526 I A*)

Hz0          92.6
B.B.G.   1.218   102.1  1.003  102.4  92.6   1.11   0.11   0.09
  "    0.1218   93.9            Q2.6   1.01   0.01   0.08

* To be described in a later publication.

The results are given in Tables III to V and in part in Fig. 2.
Titration of Type I Pneumococcus Spe$k Polysaccharide-It

will be recalled that S I is both a weak base and strong acid (9).
Since the isoeIectric substance dissolves only slowly in alkali the
titration was carried out as follows: A weighed sample of anhydrous
S I was wet with water and dissolved with a measured amount of
~/14 hydrochloric acid. The solution was then titrated back
through the isoelectric point with ~/14 sodium hydroxide.  The
finely divided precipitate dissolved readily at a reaction which was
still acid to litmus, and the titration was continued to alkalinity


134      Specific Polysaccharides

to phenolphthalein or thymolphthalein, very little additional
alkali being required between the turning points of the two indi-
cators.

0.3153 gm. of S I 58, dissolved in 10.03 cc. of ~/14 HCl, required
25.23 cc. of ~/14 NaOH (0.15 cc. being deducted as indicator
blank) to blue color to thymolphthalein, or 15.20 cc. in excess,
equivalent to 1.086 cc. of N NaOH.  Acid equivalent, 290.
0.4048 gm. of S I 59 B, dissolved in 10.03 cc. of ~/14 HCl,
required 27.28 cc. of ~/14 NaOH to bright pink to phenolphthalein,

                 0         100        zoo
I         I                 I         I     /

Fm. 2. Viscosity relations of sodium salts of specific polysaccharides.
Curve I, S III; Curve II, S I; Curve III, S. G. A. (ordinates X 0.1;
abscissra X 10); Curves IV and V, S III 0.293 and 0.0293 per cent,
respectively.

or 17.25 cc. in excess, equivalent to 1.232 cc. of N NaOH.  Acid
equivalent, 328.  Mean acid equivalent, 309.

DISCUSSION

The high values of the equivalent, conductance shown in Table I
and Fig. 1 indicate that the sodium salt of S III is a strong elec-
trolyte, since A, which is 95.7 at infinite dilution, drops to only
71.6 at a concentration of 3.85 milli-equivalents.


M. Heidelberger and F. E. Kendall

The conductivity of S III, however, does not follow the linear
relationship between A and the square root of the conqentration,
n=n,- a ~~ derived empirically by Kohlrausch and given
theoretical significance by Debye and Htickel and by Onsager (10).
In a highly ionized substance, the slope of the conductance curve,
a, depends largely on the valence type, increasing with the valence.
In the case of an electrolyte which is incompletely dissociated, such
as thallium chloride (cf. (lo)), dilution decreases the slope, owing
to the increase in the number of conductors, and the resulting
curve is concave toward the origin.  In the S III salt, however,
this effect is more than counterbalanced, and the curve becomes
convex toward the origin.  This may be explained by assuming,
with increasing dilution, the formation of ions of higher and higher
valence type as additional COONa groups dissociate. The
possibility of this taking place is shown by the structural formula
for the S III salt given below in another connection.  Thus, the
slope, a, of the conductivity curve would tend to increase with
dilution, rather than decrease, owing to the preponderating influ-
ence of the increasing valence.  That even a simpler substance
may show a similar deviation from a straight line relationship is
readily demonstrated by plotting the equivalent conductance
curves calculated from Noyes and Lombard's measurements for the
tetra and penta sodium salts of benzene pentacarboxylic acid (11).
Since the limiting conductance of the sodium ion is 50.0, that of
the Type III polycarboxylate ion (equivalent weight 340) would be
95.7 - 50.0, or 45.7, indicating that it is a ready carrier of elec-
tricity.  Its behavior is thus very different from that ascribed to a
polysaccharide anion such as that of gum arabic (equivalent weight
1200) by Taft and Malm (5), who consider the inorganic ions
responsible for the entire current carried.  The value of the limit-
ing conductance is, indeed, very close to recent values ascribed to
the caseinate (equivalent weight 2000) (12) and globulinate ions
(equivalent weight 3000) (13). The S III polycarboxylate ion,
however, is free from basic groups and has a carboxyl group for
every 340 of molecular weight.  Since all of the available evidence
indicates that there are at least eight to ten such groups in the
molecule, the cumulative effect of the negative charges on the S III
ion would be very large.  That a total charge of this magnitude
would result in large interionic or Coulomb forces can readily be
appreciated.


136      Specific Polysaccharides

Tables II and III show that although the specific viscosity,
18P = qr - 1, is independent of a 10" increase in temperature, indi-
cating that there is no association or dissociation on changing the

TABLE VI

Specific Volumes of Dissolved Svecific Polvsaccharides Accordiw to

s III

SI

s II

S.G.A.

Na benzene peg
tacarboxylat
(Noyes am
Lombard)

solvent      concen-
         tration

pa cent

Hz0         0.293
"          0.0586
d`          0.0293
`I          0.0195
"          0.01461
"          0.0117:
1.21 M KC1     0.2930
1.21 "  "      0.0293
3.35 "  "     0.2930
3.35 `I  "     0.0293
0.05 " NasHPOd  1.00
0.05 "    "    0.10
HtO         0.3273
"          0.0327
"          0.0218
L`         0.0131
1.34 M KC1     0.3273
3.35 (`  lL     0.3273
Hz0         1.106
"          0.221
0.85 M NaCl    0.553
Ha0         4.85
"         0.485
"          2.04
"          0.818
I`          0.245

3.27
1.89
1.60
1.48
1.43
1.34
1.50
1.05
1.21
1.02
4.26
1.23
2.05
1.17
1.14
1.08
1.35
1.33
1.22
1.04
1.07
1.57
1.07
1.09
1.04
1.01:

(P

-

E
7
.-

v

w cent

!3.5
L3.4
LO.0
8.4
7.7
6.3
8.7
1.1
4.2
0.44
28.3
4.6
14.9
3.5
3.0
1.8
6.6
6.2
4.4
0.87
1.6
9.6
1.6
2.0
0.87
0.33

-

lpecific
r&me

cc. Per
?a. WlU&
80
230
340
430
530
540
30
38
14
15
28
46
46
110
140
140
20
19
4
4
3
2
3
1.0
1.1
1.3

0.044
0.049
0.061

temperature in this range, aqueous solutions of S III obey neither
the Hagen-Poiseuille law, nor the Einstein relation, 7 = 90
(l++), from which `5 should equal a constant.  It is clear


M. Heidelberger and F. E. Kendall    137

(Fig. 2) that the latter function increases to a maximum with in-
creasing dilution, just as the equivalent conductance increases with
dilution. It is, therefore, possible to ascribe the increasing deviation
from the Einstein viscosity relation to the same cause; namely, the
ionization of additional COONa groups and the resultant increase in
the charge on the polycarboxylate ion.  In this instance the relation
`lap is found to hold over a limited range of concentration,
E/C

but this is not so evident in the other specific polysaccharides
studied.  Moreover, the viscosity determinations made by Noyes
and Lombard (11) on solutions of sodium benzene pentacarboxylate
show a 40 per cent increase in * with decreasing concentration

c

from 2 per cent (50 mM) to 0.25 per cent (6 mM> (Table VI).
Although this is small compared with the effect noted in the case of
S III, the negative charge due to the piling up of carboxyl groups on
even a substance of low molecular weight appears to result in a
viscosity effect in the same direction.

It is also possible to ascribe the observed viscosity effects to
hydration of the specific substance.  If this be true, the hydration
must be of a most unusual magnitude.  Otherwise, at the low con-
centrations of S III at which k'is still increasing, the concentration

c

of free water in the system would be practically constant. If
hydration be the cause of the observed anomalies, it should be
possible to calculate its extent by means of Kunitz's modification
U4), 9 = `(r'+"f)f, of Einstein's formula, and this does indeed
lead to enormous values for the specific volume in dilute aqueous
solution (Table VI), 1 gm. of S III being capable, according to the
formula, of combining with as much as 540 gm. of water.

It is also shown in Fig. 2 that if the rate of flow per second of
dilute solutions of S III in water is plotted against the pressure, a
straight line is obtained for each concentration.  These lines do not
pass through the origin, but intersect the axis at a positive value
for P which increases with dilution.  Thus a certain pressure must
be exceeded before flow starts, and the solution behaves as a plastic
solid.  This might be accounted for by assuming a definite orien-
tation of the highIy charged polycarboxylate ion at high dilutions


138      Specific Polysaccharides

at which dissociation is approaching completion.  This would also
entail an orientation of the accompanying sodium ions, and a
definite structure would result, somewhat as follows:

in which RCOO- represents the glycuronic acid residue in gluco-
sidic union with the glucose residues (cf. (3)). Such an arrangement
should also result in a maximum value of % and is consistent with
the explanation given for the positive deviation from the square
root conductivity relation at high dilutions.

In the case of the specific polysaccharide of Type I pneumo-
coccus the viscosity anomalies are smaller than in the case of S III,
and this might be ascribed either to the smaller magnitude of the
Coulomb forces engendered by the S I anion, or to the lower extent
of the hydration,  The acid equivalent of S I is somewhat lower
than that of S III, but since there is one basic group for approxi-
mately every three carboxyl groups, the total negative charge on
the S I ion would be less than that of S III, assuming the molecular
weights to be approximately the same.  Also, calculation of the
hydration by Kunitz's formula yields lower values than in the case
of S III, so that either explanation might be valid.

In the case of S II and the specific gum arabic, with their acid
equivalents of approximately 1000, both viscosity and hydration
are low. Since there is an apparent parallel between the total
acidity of the specific polysaccharides considered and their hydra-
tion as calcuIated by the Kunitz formula, it is possible that the
unusual hydration, or at least the unusual viscosity, of the S III and
S I is conditioned by the unusual magnitude of the total negative
charge on these ions.

Association of abnormally high viscosities with an acid poly-
saccharide has been shown qualitatively by Raistrick and Rintoul
(15) in the case of luteic acid, a polysaccharide produced from


M. Heidelberger and F. E. Kendall     139

glucose by PeniciUium luteurn, Zukal. The structural unit is
composed of 2 molecules of glucose and 1 of malonic acid, one
of the carboxyl groups being free. Moreover, Staudinger and
Kohlschiitter (16) have recently continued earlier work by
Staudinger and Urech (17), giving preliminary viscosity data on
polyacrylic acids. They fix the relative viscosity of a 0.1
equivalent solution of a sodium salt at the enormous value of
114 and the relation F at about 1140, values far higher than
obtained in the case of S III.  The polyacrylic salts differ from
those of S III, however, in hydrolyzing readily and in the smaller
equivalent conductivity of their aqueous solutions. Staudinger
originally considered the high viscosity of polyacrylic acid salts to
be due to hydration caused by the high ionic charge, but now
ascribes it to "swarms" caused by increasing ionization.  If, as
seems agreed, these high viscosities are conditioned by the piling
up of large Coulomb forces, regardless of the mechanism by which
this is brought about, a substance such as acrylic acid, with an
acid equivalent of only 86 would be expected to develop higher
viscosities as the chain of carbon atoms and carboxyl groups is
lengthened by polymerization, than would the aldobionic acid
structural unit of S III, with its acid equivalent of 340.

Tables II and III show clearly the effect of electrolytes in dimin-
ishing the viscosity (and in Table VI the hydration) of the solutions
of S III and in bringing them into agreement with the Hagen-
Poiseuille law and the Einstein equation.

It is shown in Table IV that F increases to a maximum with
increasing dilution in the case of the sodium salt of S I as in the
case of S III, but that the increase is much smaller, and that z
                             Y'C
is constant over a very limited range.  Although titration shows
that the acid equivalent of S I is lower than that of S III, the
greater number of carboxyl groups in the molecule and their
expected effect on the total charge and viscosity appear to be more
than negatived by the simultaneous presence of basic groups.
However, the internal compensation of Coulomb forces is insuffi-
cient to prevent the building up of a negative charge large enough
to produce viscosity effects qualitatively similar to those encoun-


140      Specific Polysaccharides

tered in the case of S III (see also Fig. 2) ; and in S I, also, these
forces are only incompletely reduced by isotonic concentrations of
salts.  The smaller viscosity effects might also be attributed to a
lower molecular weight for S I than for S III, but no evidence is
available on this point.  Although the total viscosity effects of the
interionic forces due to the S I anion do not reach the proportions
shown by the S III ion, when these forces are depressed by large
concentrations of salts or acids, the specific viscosity of S I is very
close to that of S III.  This is perhaps a reflection of similarities in
the arrangement of the sugar and sugar acid units of which the two
substances are composed.

The low acid equivalent of S I may be taken as evidence that
some other explanation must be sought for the widely differing
ratios between precipitin index and mouse protection in the case
of Type I and Type III antisera than the suggestion of Sobotka and
Friedlander (18) that the ratios are connected with differences in
the acid equivalents of the two polysaccharides.

Table V shows that in the non-basic specific gum arabic (19)
(S.G.A., acid equivalent 835) the accumulation of negative charges
is still great enough to give rise to an increase in %' on dilution,
                             c
while this effect is lost in the case of the specific polysaccharide of
Type II pneumococcus (9) (S II, acid equivalent 1250). The
specific viscosities of these polysacchsrides are also lower than
those found at similar concentrations of S I and S III, but are
higher than that of a non-acidic (and non-basic) bovine tubercle
bacillus polysaccharide (B.B.G., Table V).

In the series of specific polysaccharides studied, therefore, there
appears to be a relation between the magnitude of the viscosity
phenomena shown by the substance and the frequency of occur-
rence of carboxyl groups in the polysaccharide chain.  That the
large viscosity effects exhibited by salts of S III are due to the con-
sequent piling up of negative charges in the salts of this strong
multivalent acid is believed to be indicated by the data presented.

BUMMARY

1. The specific polysaccharide of Type III pneumococcus is
shown to yield a highly ionized sodium salt characterized by a
mobile negative ion of very high valence.


M. Heidelberger and F. E. Kendall

2. Viscosities have been determined for a number of specific
polysaccharides under varying conditions of salt concentration,
and the findings correlated with the magnitude of the charge on the
anion.

3. So strong are the interionic, or Coulomb, forces engendered
by the S III polycarboxylate ion that the ratio `$' increases many
times with dilution.  At increasing dilutions S III solutions show
in increasing measure the yield point phenomenon characteristic of
plastic solids, an effect .possibly due to the orientation of ions
brought about by these forces.

4. The specific polysaccharide of Type I pneumococcus shows
viscosity abnormalities of the same character, but of smaller
magnitude.  Although its acid equivalent is even lower than that
of S III, internal compensation of negative charges by the basic
groups present is believed to be the cause of the smaller effect.
5. The data on the specific gum arabic and Type II pneumo-
coccus polysaccharide show that the viscosity effects decrease with
the relative number of carboxyl groups, or negative charges, in the
molecule.

BIBLIOGRAPHY

1. Cj. Heidelberger, M., Physiol. Reu., 7,107 (1927) for a review.
2. Heidelberger, M., and Goebel, W. F., J. Biol. Chem., 70,613 (1926).
3. Heidelberger, M., and Goebel, W. F., J. Biol. Chem., 74,613 (1927).
4. Thomas, A. W., and Murray, H. A., Jr., J. Physic. Chem., 33,676 (1928).
5. Taft, R., and &Ialm, L. E., J. Physic. Chem., 36,874 (1931).
6. Shedlovsky, T., J. Am. Chem. Sot., 62,1793 (1930).
7. Shedlovsky, T., J. Am. Chem. Sot., in press.
8. Bingham, E. C., Fluidity and plasticity, New York (1922).
9. Heidelberger, M., Goebel, W. F., and Avery, 0. T., J. Exp. Med., 42,
  727 (1925).

10. Onsager, L., Physik. Z., 27,388 (1926); 28,277 (1927).
11. Noyes, A. A., and Lombard, R. H., J. Am. Chem. Sot., 33,1423 (1911).
12. Greenberg, D. M., and Schmidt, C. L. A., J. Gen. Physiol., 7, 287, 303,

317 (1924-25).

13. Spiegel-Adolf, M., Die Globuline, Dresden (1930).
14. Kunits, M., J. Gen. Physiol., 9, 715 (1925-26).
15. Raistrick, H., and Rintoul, hi. L., Phil. Tr. Roy. Sot. London, Series B,

220, 255 ( 1931).

16. Staudinger, H., and Kohlschiitter, H. W., Ber. them. Ges., 64,209l (1931).
17. Staudinger, H , and Urech, E., Helv. chim. acta, 12,1107 (1929).


142      Specific Polysaccharides

18. Sobotka, H., and Friedlander, M., J. Ezp. Med., 63,299 (1931).  Fried-
  lander, M., Sobotka, H., and Banahaf, E. J., J. Exp. Med., 47, 79
  (1928).

19. Heidelberger, M., Avery, 0. T., and Goebel, W. F., J. Exp. Med., 49,
  347 (1929). Heidelberger, M., andKendall, F. E., J. Biol. Chena., 84,
  639 (1929).