THE SOLUBLE SPECIFIC SUBSTANCE OF
        PNEUMOCOCCUS.

THIRD PAPER.

BY MICHAEL HEIDELBERGER, PH.D., WALTHER F. GOEBEL, PH.D., AND
             OSWALD T. AVERY, M.D.

(From tke Hospital of The Rockefeller Institute for Me&al Research.)

(Received for publication, June 30, 1925.)

In the preceding papers of this series (1) the significance of the so
called "soluble specihc substance" of Pneumococcus (2) was discussed
and methods were given for the isolation of the specific substances of
Types II and III pneumococcus. In the present communication
refmements of these methods are described. With their aid it
has been possible to obtain both of these substances free from nitrogen
and possessing much greater activity when tested with the homologous
immune sera than did the products previously isolated. Further
data are presented concerning the nature of the polysaccharides
with which these substances appear to be identified.

The isolation of a specific substance from Type I pneumococcus is
also described, and it is shown that this substance, while apparently
also a sugar derivative, contains nitrogen and differ also in other
respects from the specific substances isolated from the other two
fixed types of pneumococci.

EXPER&ENTAL.

  1. The Soluble Specijic Substance of Type I Pnemwcoccus.
The original method of purification of the soluble specific substance
was modified after numerous trials to fit the properties of the Type I
substance.

8 day cultures of Type I pneumococcus in meat infusion phosphate broth are
concentrated in 21 liter lots on the water bath to a volume of 1.2 to 1.5 liters.
Tbe concentrate is precipitated with 1.4 volumes of alcohol and put through tbe
                      727


728     SPECIFIC SUBSTANCE OF PNEUMOCOCCUS.   PAPER III

initial 3 layer separation as described in Paper I.  The middle layer' is dissolved
as well as possible in 200 to 300 cc. of water and the mixture is centrifuged and
the precipitate washed twice with small amounts of water.  The clear solution
and washings are kept on ice and combined with subsequent lots until 300 to 325
liters have been worked up. After solution of the deposit of salts the total
concentrate, at a volume of about 4.5 to 5.0 liters, is treated with 1:l hydro-
chloric acid until strongly acid to Congo red. The resulting precipitate is
allowed to settle for several hours in the cold, after which as much of the super-
natant liquid as possible is siphoned off and the remainder cleared by centrifuga-
tion. The precipitate is washed once with 0.01 normal hydrochloric acid and
again separated by repeated centrifugation at high speed. The clear solution
and washings, at a volume of 5 to 6 liters, are now precipitated with 7 to 8 liters
of chilled alcohol and allowed to stand overnight in the cold.  A test portion of
the supematant liquid, when neutralized and boiled down to small volume,
should give no immediate precipitate with Type I antiserum. As much as
possible is siphoned off and the crude speci6c substance separated from the
remainder by centrifugation.  The precipitate is then washed in the centrifuge
bottles with 0.5 normal acetic acid, in which the isoelectric Type I substance is
insoluble, while most of the accompanying glycogen or erythrodextrin dissolves.
After several hours in the cold the mixture is centrifuged and the precipitate
taken up in water. The specific substance is dissolved by addition of sodium
hydroxide until the mixture is faintly alkaline to litmus, the volume is adjusted
to about 700 cc., and the mixture is centrifuged at high speed until clear.  The
precipitate is washed with 50 cc. of water and again centrifuged.  To the solu-
tion and washings are added 20 gm. of sodium acetate, and when this is dis-
solved 400 cc. of alcohol are added, with vigorous stirring, and the mixture is
allowed to stand overnight in the cold.  In relatively concentrated solution and
in the presence of sufficient salts the Type I specific substance is quantitatively
precipitated by 0.5 volume of alcohol in the cold. The use of sodium acetate
is advantageous as it is not thrown out by alcohol at the concentrations used.
The precipitate is centrifuged off, water is added up to a volume of about 500
cc., the mixture is made slightly alkaline again if necessary, and is finally centri-
fuged to remove any insoluble material.  The solution is then stirred mechan-
ically and the specitic substance is rendered insoluble by the careful addition of
16 cc. of glacial acetic acid. After several hours in the cold it is centrifuged off
and redissolved with the aid of sodium hydroxide as before, except that the
volume should now be about 400 cc.  15 gm. of sodium acetate are added, and
the specific substance is precipitated in the cold with 200 cc. of alcohol.  After
having stood overnight it is centrifuged off and redissolved in enough water to
bring the volume to about 250 cc., using enough sodium hydroxide to make

t Centrifuged precipitates containing the soluble specific substances are
usually compact and gummy, and care must be taken to smooth out all lumps
when the deposit is redissolved.


M. HEIDELBERGER, W. P. GOEBEL, AND 0. T. AVERY   729

the reaction slightly alkaline. The solution is chilled, stirred mechanically,
and treated with an equal volume of barium hydroxide solution in equilibrium
with the crystalline phase at about 50'.  Most of the specific substance separates
at once, and the mixture may be centrifuged after several hours in the ice box.
Under optimal conditions the precipitation is complete, but occasionally up to
about 15 per cent of the specific substance may remain in solution.  If this is to
be recovered it is best worked up separately, as coloring matter and other im-
purities are eliminated with it. The barium hydroxide precipitate is taken
up in about 400 cc. of water and treated with 5 normal sulfuric acid until, after
all lumps are smoothed out, the reaction remains acid to Congo red.  The mix-
ture is then centrifuged, and the precipitate of barium sulfate washed twice
with very dilute sulfuric acid. The supematant solutions are neutralized, and
if opalescent, may be passed first through a Berkefeld V candle and then through
the W grade. The solution is concentrated in O~GUO to about 200 cc., made
faintly alkaline with sodium hydroxide, and precipitated in the cold with 100 CC.
of alcohol after the addition of 10 gm. of sodium acetate. The next day the
precipitate is collected in the usual way, redissolved in water, and at a volume
of about 200 cc. is again thrown out at the isoelectric point with 7 cc. of glacial
acetic acid, using the same precautions as before. The precipitate is taken up
in a little water and redissolved by addition of 1 :l hydrochloric acid.  The vol-
ume is made up to about 125 cc. with water and hydrochloric acid, and the
speciiic substance is then precipitated as the hydrochloride by the addition, with
mechanical stirring, of 250 cc. of chilled redistilled alcohol. The centrifuged
product is redissolved and reprecipitated in the same way and is then taken
up in as small a volume as possible of cold water.  The extremely viscous solu-
tion is then dialyzed in collodion bags in the ice box against successive changes
of distilled water. The Type I specific substance is a weak base, and as the
excess of hydrochloric acid diffuses out the hydrochloride is hydrolyzed and the
isoelectric substance precipitates. Completion of the process and removal of
the last traces of chlorine ion are accelerated by miximg the contents of the bags
each time the water is changed. The mixture is finally centrifuged, the soIu-
tion, which contains a little active material, is added to the next preparation,
and the precipitate is washed with acetone, tiltered on a hardened paper in a
Biichner funnel, and dried. The yield should be from 2 to 3 gm.

The Type I specific substance obtained by this method contains
5 per cent of nitrogen and is often ash-free and colorless, although some
preparations are still grayish in color and contain a little ash.  In its
isoelectric form the product is very sparingly soluble, but may be
dissolved with the aid either of alkali or mineral acid.  Concentrated
solutions are extremely viscous. The specific optical rotation, +300",
is about the same on either side of the isoelectric point, which is


730     SPECIFIC SUBSTANCE OF PNEUMOCOCCTJS. PAPER III

in the vicinity of pH 4. When boiled with a sufficient excess of
mineral acid the substance is slowly hydrolyzed with formation of
reducing sugars, which it has not yet been possible to identify fully.
Reducing sugars also appear when the substance is treated with
nitrous acid in the cold. One-half of the nitrogen is evolved at the
same time, with simultaneous disappearance of the specific reaction.
Since the specific substances of Types II and III pneumococcus are
unaffected by nitrous acid under these conditions, it would appear
that this half of the nitrogen, at least, is an integral part of the specific
substance and is either linked to the reducing group of a sugar deriva-
tive, as Karrer believes is the case with the polyglucosamines (3), or
else is so placed in the complex molecule that its removal causes some
other type of scission. About one-half of the remaining nitrogen in
the substance is given off within 15 minutes when the reaction mixture,
freed from nitrous acid, is made alkaline and distilled with steam.
The substance contains no sulfur or phosphorus.

When a concentrated aqueous solution of the hydrochloride of the
specific substance is carefully treated with 1:l hydrochloric acid a
point is reached, at an acidity of 1.3 normal, at which the substance
again precipitates, redissolving again if still more acid is added.
Whether this indicates lactam formation, or some other reversible
intramolecular change, is not yet clear.

3 per cent solutions, prepared by dissolving the specific substance
in dilute acid and bringing it back past the isoelectric point with dilute
sodium hydroxide, give precipitates with solutions of the following
reagents: barium hydroxide, copper sulfate, silver nitrate, neutral
and basic lead acetates, uranyl nitrate, and phosphotungstic acid.
There is no color developed with iodine-potassium iodide solution,
nor does the unhydrolyzed substance show reduction with Fehling's
solution. The biuret, tannic acid, xanthroproteic, and ninhydrin
tests are negative. With Millon's reagent a jelly is formed in the
cold, but the precipitate redissolves on heating and no color is
developed. Potassium pennanganate is not immediately reduced.
After hydrolysis with nitric acid and subsequent oxidation mucic

acid was obtained.

1.5 gm. of air-dry Preparation 39 were dissolved in 200 CC. of hot 1.4 normal
nitric acid and boiled under a reflux condenser for 4 hours.  The resulting green-


M. HEIDELBEBGER, W. F. GOEBEL, AND 0. T. AVERY    731

ish yellow solution was concentrated in wacuo to about 10 cc., and 4 cc. of con-
centrated nitric acid were added. The next day the mixture was boiled for a
few moments and then stirred on a large clock-glass over boiling water until
dry.  The process was repeated with a few cc. of 1:l nitric acid, after which the
dry, white residue was taken up in acetone and allowed to stand overnight.
The crystalline residue was filtered off, washed with a little acetone, and taken
up in 5 cc. of normal ammonium hydroxide.  The insoluble portion, apparently
calcium oxalate, was discarded.  Addition of 0.3 cc. of concentrated nitric acid
to the titrate resulted in the deposition of 0.21 gm. of mucic acid melting and
decomposing at 216' with preliminary softening and blackening. The acetone
mother liquors above were concentrated to dryness and taken up in water, when
a second fraction of muck acid was left behind.  This was purified in the same
way, yielding 0.07 gm., melting and decomposing at 215".  For analysis the two
fractions were combined and recrystallized from water.

0.1008 gm., anhydrous, ash-free substance gave 0.1253 gm; CO2 and 0.0442
gm. H,O.

Calculated for CeHr&a: C, 34.27 per cent; H, 4.80 per cent.  Found: C, 33.9
per cent; H, 4.9 per cent.

From the aqueous mother liquors from which the second crop of mtic acid
was obtained was isolated an acid potassium salt in a yield of 0.191 gm. and
containing 22.3 per cent of potassium.

A repetition of the oxidation in the cold with 1 gm. of Preparation 41 gave
after 1 week a deposit of mucic acid weighing 0.115 gm. after two recrystalli-
xations.

Since the Type I specific substance gives a positive test for glu-
curonic acid with naphthoresorcinol it is possible that galacturonic
acid is present as a part of the molecule, for this would yield muck
acid on oxidation.  That the mu& acid does not arise from oxidation
of galactose itself is indicated by the difliculty with which an osazone
is formed from the products of hydrolysis, although crystallization of
the osazone formed might also be hindered by the complexity of the
reaction mixture.

While the Type I specific substance as at present obtained probably
does not represent a single, chemically pure compound, it would never-
theless seem as if the major portion of the impurities had been
removed.  As will be seen from Table I, in which the figures given
are calculated to the ash-free basis, the total nitrogen, the nitrogen
and reducing sugars liberated on treatment with nitrous acid, and the
optical rotation are remarkably constant in the majority of instances,
although the preparations listed were isolated in different ways.


732     SPECIFIC SUBSTANCE OF PNEUMOCOCCUS. PAPER III

29B was prepared from Type I pneumococci themselves, instead of
the autolyzed culture fluid, by solution in diluted bile, removal of
the "nucleoprotein" with acetic acid, and adsorption of the specific
substance on alumina, as described in the case of Type II pneumococ-
cus (1).

In 36 and 37 the use of barium hydroxide was omitted, necessitat-
ing a more laborious fractionation. 37A was prepared from 37 by
adsorption on a particularly active form of alumina (Alumina A)
prepared according to the directions of Willstatter and Kraut (4).

41B3 was obtained from 41 by interaction with neutralized formalin
in slightly alkaline solution.  Discharge of the phenolphthalein color
indicated that the specific substance combined with the aldehyde.
However, fractional precipitation of the resulting substance with
alcohol (Bz being the second fraction) failed to yield a product with
different properties. That the combination with formaldehyde was
a labile one was indicated by the fact that the product recovered,
when once more treated as before with formalin, again showed an
increase in the acid reaction, indicating that any formaldehyde com-
bined with the specific substance had been eliminated in the process of
isolation (addition of cold dilute hydrochloric acid and precipitation
with alcohol).

43A was obtained by digestion of the product of the third alcoholic
precipitation (see method) with an amount of ice-cold water sufIicient
to dissolve only a portion of the spe&c substance.  The mixture had
a total volume of 300 cc. in this case, and the specific substance was
reprecipitated with alcohol and then purified with the aid of barium
hydroxide.  The residue from the A fraction was worked up in the
usual way as 43B, while the part of it not precipitated by barium
hydroxide, but separating when the alkaline mother liquors were acidi-
fied with acetic acid, is represented by 43C.  It will be seen from Table
I that each of these fractions gave the same analytical values, and the
physical properties corresponded equally closely.

Finally, 44A, after precipitation with barium hydroxide, removal
of the barium, and precipitation with alcohol, was purified twice over
the sparingly soluble hydrochloride mentioned above.  It was hoped
that precipitation in this way by hydrochloric acid without the assist-
ance of alcohol would result in the removal of nitrogenous or carbo-


Y. HEIDELBEBGER, W. F. GOEBEL, AND 0. T. AVERY   733

TABLE I.

              C    H   Ash.

                                 -
Type I pneumococcus.

29B    +3of
36    +304sc
37    +295 .5c
37A   $287'
38A   +310o
39    +304O
41    +303O
41B,   +279O
#A   +303O
43B    +300o
43c   +300o
44A   +300o

2s     +63.2' 0.18
2SA    +70.2' 0.0
25B    +56.7' 0.0
25c    +72.2' 0.12
26A    +!5.2O 0.0
26A,    +72.8O 0.0
268    +54.4O

br `ml
4.8
4.6:
5.1
4.11
5.0
4.41
5.0
4.43
5.0
4.9
5.0
5.0

   -
LLwCmi
2.7
2.4
2.6
2.5
2.6
2.5
2.6
2.5
2.5
2.5
2.6
2.6
   -

A
MI cnrl
31.8
31.6
27.2

28.6

28.4

-

31.7
28.5
27.1
26.0
23.9
28.8
28.5
28.0

28.5 43.3 5.5

Type II pneumococcus.

80.3
68.4

67.6
69.1
68.2
56.0

`acud
5.8

1.5
0.7
7.9
1.8
1.6
0.0

1:4,000,000
1:6,000,000
1:6,000,000
1:6,000,00@
1:6,000,000
1:6,ooO,OOO
1:8,000,000

1.2  1:6,000,000
0.0  1:6,000,000
0.0  1:6,000,000
0.5  1:8,000,000

1302  45.8
946
1190
1258
1252
1105

   1.9
6.4' 0.35

1.6
0.0

1:3,000,000
1:6,000,000
1:2,000,000
1:5,ooo,ooo
1:6,000,000
1:5,000,000

Type III pneumococcus.

30     -37.3O          73.0   330             1:6,000,000
301    -30.9O  0.0      73.3   347            1:5,000,000
30A    -35.P  0.0       71.0   339          0.0  1:6,000,000
33     -3oY  0.0       73.0   343          0.0
331    -32.5'  0.0      74.5   341          0.0
33rI    -34.0*  0.0       75.5   340  42.7   5.3
33B III   -30.8'  0.0       72.5   358          0.0  1:6,000,000

* Calculated as glucose.
t 2 hours at 37' and overnight in the cold.
$ Micro Kjeldahl determinations using aeration method for collecting NI&.
Results about 10 per cent low.  In the other cases hegl's method wzs used.


734     SPECIFIC SUBSTANCE OF PNEUMOCOCCUS. PAPER III

hydrate material that would otherwise be precipitated by the alcohol
customarily added.  However, this preparation checked closely with
the others in optical rotation, carbon, hydrogen, and nitrogen, and
in the other quantitative and qualitative tests used.

2. Type II Pneunwcoccus.

A. Further Purification of the Type II Soluble Speci$c Substance.

The spechic substance from 312 liters of broth culture was isolated according
to the method given in Paper II.  After the thiid precipitation with ammonium
sulfate the deposited material was redissolved in hot water, run through a Berke-
feld W candle to remove opalescence, and concentrated to 150 cc. The solu-
tion was cooled to 0", acidified with 35 cc. of 1:l hydrochloric acid, stirred
mechanically, and precipitated by the addition of 350 cc. of chilled redistilled
alcohol.  After 2 hours in the ice box the precipitate was centrifuged off in the
cold and redissolved in 75 cc. of cold water. The precipitation process was
then repeated with 18 cc. of 1: 1 hydrochloric acid and 300 cc. of alcohol.  The
precipitate, after centrifuging, was redissolved in 100 cc. of water, rinsed into
a collodion bag with 5 cc. of normal hydrochloric acid, and dialyzed in the cold
against distilled water until free from chlorine ion. The solution was concen-
trated to 50 cc. in uocz(o and stirred into 600 cc. of redistilled acetone.  After
having stood overnight the precipitate was collected on hardened paper, washed
with redistilled acetone, and dried.  The yield was 3.3 gm. in the case of Prepara-
tion 26A, and 2.4 gm. for 25A.

The supernatant from the initial acid-alcohol precipitation still reacted strongly
with Type II antiserum and was accordingly precipitated with 0.5 volume of
ether.  The material deposited was dissolved in 20 cc. of water and, after addi-
tion of a little hydrochloric acid, was dialyzed as in the previous instance, con-
centrated to 20 cc., and poured into 12 volumes of acetone.  0.6 gm. was recov-
ered (26B) in one case and 0.5 in another (25B).

In an attempt to push the purification further a portion of 26A was again put
through the acid-alcohol precipitation process. The product, 26A1, gave,
however, the same analytical values as that from which it was derived.

From a comparison of Preparation 2.5, Table I, in which these addi-
tional steps in the purification process were omitted, with 25A, 26A,
and 26A1, it will be seen that the remaining nitrogen in the specific
substance has been eliminated, all but traces of ash have been
removed, a fraction (25B and 26B) has been separated containing
impurities of lower optical rotation, and the highest dilution at which
the substance reacts with its homologous serum has been increased.
In order to reduce the analytical error to a minimum micro Kjeldahl
determinations were run on 25 mg. of substance in the case of 26A.


M. HEIDELBERGER, W. P. GOEBEL, AND 0. T. AVERY   735

B. Cm..rmatimz of @ucose as the Chief Sugar Constituent of the
         Type II Specif;c Substance.

In the previous papers it was shown that glucosazone could be
obtained from the hydrolysis products of the Type II soluble specific
substance. Although the hydrolyzed material showed a specific
rotation of about +55", indicating that the resulting hexose was
probably glucose, the possibility that the sugar might have been man-
nose or fructose was by no means excluded, `owing to the possible
presence of other optically active substances.
In order detiitely to determine this point, a portion of the specific
substance was hydrolyzed and subsequently oxidized.

0.5 gm. of Preparation 25A was dissolved in 25 cc. of water and boiled for 5
hours under a reflux condenser with an equal voIume of normal nitric acid.  The
solution was evaporated in vacua to 3 to 4 cc., treated with 2 cc. of concentrated
nitric acid, and allowed to stand at room temperature overnight.  The mixture
was then boiled for 2.5 minutes over a tlame, and poured on to a large watch-
glass, stirred, and quickly evaporated over a boiling water bath.  A thick paste
was obtained and thii was twice evaporated with water to expel the last traces
of nitric acid, dissolved in 2 cc. of water, made strongly alkaline with 40 per
cent potassium hydroxide solution, and reacidifkd with glacial acetic acid.
The mixture was cooled in ice water, and after 24 hours in the cold the crystals of
potassium acid saccharate which had separated were filtered off. 0.1 gm. was
recovered. This was recrystallixed from 1.5 cc. of water, yielding 0.062 gm.
of the purified salt.
0.0602 gm. substance gave 0.0210 gm. RsSO4.
Calculated for KOOC(CHOH)~COOH: K, 15.75 per cent. Found: 15.65
per cent.

The isolation of potassium acid saccharate through the oxidation of
the hydrolytic products of the Type II soluble specific substance,
together with the isolation of glucosazone previously reported, leaves
little doubt that the units from which the polysaccharide is built up
are those of glucose.

C. Preparatiole of a Triacetute of the Ty$e II Soluble Specific
             Substance.

If the true soluble specific substance were actually a polysaccharide
it should be possible to convert it into an acetyl derivative just as
the dhlo~es and starches may be acetylated.  On the other hand,


736    SPECIFIC SUBSTANCE OF PNEUMOCOCCUS. PAPER III

if the polysaccharide present were an impurity the altered solubilities
of an acetyl derivative might be expected to facilitate its removal
from the actual specific substance.

0.5 gm. of Preparation 25A was accordingly dried to constant weight in oacw,
and shaken overnight at room temperature with a mixture of 2.5 cc. of dry acetic
anhydride and 6 cc. of dry pyridine.  Since no reaction appeared to have taken
place the tightly stoppered bottle containing the mixture was heated in a water
oven for 24 hours. The resulting suspension, which had darkened somewhat,
was poured into ice water but most of the solid failed to dissolve.  It was there-
fore titered off, macerated with water until the washings failed to give a spetic
test with Type II antiserum, and shaken with 200 cc. of water and a few glass
beads until it had disintegrated into a very fine powder.  After centrifugation
the supematant fluid gave only a weak test with immune serum.  The residue
was next washed with 50 cc. of methyl alcohol, which removed most of the
coloring matter, and was dried in vacz4o. 0.7 gm. of an amorphous, fluffy, tan
powder was recovered.

0.1009 gm. substance gave 0.1864 gm. CO2 and 0.0503 gm. HsO.
Calculated for [C&I&(OCOCH&: C, 49.95 per cent; H, 5.60 per cent.
Found: C, SO.4 per cent; H, 5.6 per cent.
0.1084, 0.1190 gm. substance hydrolyzed with OS N NaOH, neutralized 9.70,
10.90 cc. 0.1 N NaOH respectively.  Calculated: 11.30, 12.40.
The triacetate was found to be practically insoluble in water and the
common organic solvents.  It dissolved, however, when warmed with
dilute sodium hydroxide, the neutralized solution giving a heavy
precipitate with antiserum.  On account of the extreme insolubility
of the triacetate it was difficult-to determine definitely whether or not
it was specific in itself.  However, the large yield and the fact that it
again yields a specifically reacting substance on hydrolysis showed that
no separation of the polysaccharide from the actual specific substance
had been effected.

D. Recovery of the Type II Specific Szlbstance from Its Precipitate with
          %omologous Antibody.

In Paper II it was shown that the Type II specific substance could
be recovered from its precipitate with immune serum. Owing to the
difliculty met with in separating the polysaccharide from large amounts
of serum protein it seemed advisable to repeat this experiment, using
instead of the serum an antibody solution purified essentially accord-
ing to Felton (5).


Y. HEIDELBERGER, W. P. GOEBEL, AND 0. T. AVERY   737

1.5 liters of Type II antipneumococcus serum were gradually added with stir-
ring to 28.5 liters of well chilled ~/lo0 acetic acid-sodium acetate buffer at pH 5.
After having stood overnight the precipitate was centrifuged off, dissolved in
750 cc. of 0.85 per cent salt solution, centrifuged once more, and again added to
19 volumes of the same buffer solution.  The resulting precipitate, which con-
tained a large proportion of the antibodies in the original serum, was finally
collected as before and dissolved in 1.5 liters of saline.

To this solution was slowly added 0.2 gm. of Preparation 25.4 dissolved in 500
cc. of saline.  After 2 hours in the incubator at 37" and standing in the ice box
overnight the precipitate was centrifuged off and was washed with 150 cc. of
saline, centrifuged again, and suspended in 150 cc. of a citrate-phosphate-borate
buffer solution prepared according to Northrop (6).  The mixture was digested
at 37' with 1 gm. of trypsin added in small portions at intervals until a clear
solution was obtained.  This was then treated with 2 volumes of alcohol and the
resulting precfpitate was suspended in water and treated with dilute sodium
hydroxide solution until it just dissolved. The solution was heated to boiling
and neutralized with dilute acetic acid, causing the separation of a coagulum.
The supematant liquid containing the specific substance was evaporated to
complete dryness and taken up in boiling water.  A small amount of insoluble
protein material was centrifuged off and the supematant liquid was dialyzed
until free from salts and repeatedly evaporated and extracted with water.  With
each evaporation a small amount of insoluble protein material was eliminated.
The extract was finally saturated with ammonium sulfate, whereby a heavy
yellow protein precipitate separated.  This was centrifuged off, redissolved in
a little water, reprecipitated with ammonium sulfate, and again centrifuged.
The supematant liquid containing the soluble substance was again dialyzed
until free from sulfate. The solution was then concentrated to 5 cc. and pre-
cipitated with 10 cc. of alcohol and a few aystals of sodium acetate.  The
inactive supematant liquid was discarded and the precipitate was twice repre-
eipitated by alcohol from a volume of 5 cc.  The substance was finally precipi-
tated from the same volume at OS by the addition of 1.5 cc. of 1:l hydrochloric
acid and 2 volumes of alcohol.  It was then dissolved in a few cc. of water and
added to a large excess of acetone.  The yield was 0.12 gm.

The recovered specific substance (2X) failed to give the biuret
reaction and contained only 0.12 per cent of nitrogen, whereas in the
original experiment with serum the nitrogen content was 1 .O per cent.
In its other properties it agreed well with the highly purified material
used to precipitate the antibody solution.


738     SPECLFIC SUBSTANCE OF PNEUMOCOCCUS. PAPER III

3. The Soluble Specijic Substance of Type III Pmumococcus.
     A. Further Attempts at Purification,

A preparation of the Type III specific substance which had been
twice putied by precipitation with hydrochloric acid (see Paper II)
was reprecipitated three times in this manner in order to determine
whether any additional impurities could be eliminated. However,
the preparations, 33,331, and 3311 checked well in analytical values
with each other and with previous preparations, except that no nitro-
gen was found, although in the case of 33135.5 mg. of substance were
used for each micro Kjeldahl determination.

Since it had been found that the Type III substance gave a precipi-
tate when barium hydroxide in excess was added, an additional puri-
fication of the substance was attempted over the barium &It.

0.5 gm. of Preparation 33 was 6nely pulverized and suspended in 25 cc. of
water.  The mixture was heated to boiling under a reflux condenser and to it
was added enough 0.2 normal barium hydroxide solution to keep the reaction
just alkaline to phenolphthalein. The solution was centrifuged from a few
particles which had failed to dissolve, and the supernatant liquid, which con-
tained practically all of the substance as a soluble barium salt, was treated with
a slight excess of saturated barium hydroxide solution. After the resulting
heavy flocculent precipitate was centrifuged off the supernatant liquid gave
only a slight precipitate with immune serum. The barium salt was washed
once with water containing a small amount of barium hydroxide and was then
dissolved in about 350 cc. of hot water and treated cautiously with sulfuric acid
until the supematant liquid showed neither barium nor sulfate ions.  The barium
sulfate was centrifuged off and the supernatant liquid concentrated in wcuo to
small bulk and dialyzed in a collodion bag.  After further concentration in
uucuo the solution was added to 10 volumes of redistilled acetone. The pre-
cipitate was sucked dry on a filter, washed with acetone, and dried in wacuo
over phosphorus pentoxide until constant in weight, a process which rendered
the recovered Type III acid insoluble.  The yield was 0.43 gm.
A comparison of the properties of Product 33B III with those of

33 (Table I) indicates that precipitation of the Type III acid as the
barium salt does not effect a fractionation.

An attempt was also made as in the case of Type I to remove possible
impurities by adsorption of the specific substance on the highly reac-
tive Type A alumina prepared according to the method of Willstftter
and Kraut (4),


M. HEIDELBERGER, W. F. GOEBEL, AND 0. T. AVERY    739

As a result of preliminary experiments it was found that at a pH below 6.0
1 gm. of gel (calculated as Al&a) adsorbed approximately 0.35 gm. of polysac-
&ride. At a pH higher than 7.0 the adsorption of the Type III substance
did not reach completion.

0.5 gm. of Preparation 30 was dissolved in the equivalent amount of N/14
sodium hydroxide.  To the solution was added a suspension of 1.5 gm. of alumina
and the volume was made up to 750 cc.  The pH was adjusted with N/5 hydro-
chloric acid to 5.0 and the suspension was shaken for 1 hour, after which the
supernatant fluid gave a negative precipitin test.  The mixture was centrifuged
and the precipitate washed once with water.  It was then extracted twice with
N/S sodium carbonate solution and the extract, after neutralization, was con-
centrated in UUCUO, dialyzed in parchment, again concentrated in YUCUO, and
dialyzed in collodion until free from chloride ion.  The solution was then further
concentrated in wcuo and precipitated from a volume of 15 cc. by 5 cc. of 1: 3
hydrochloric acid and 40 cc. of redistilled alcohol at 0".  The specik acid was
washed free from chlorides with 50 per cent alcohol and finally with acetone.
0.36 gm. of dry material was recovered.

A comparison of Product 30A with the starting material shows
that adsorption of the specific substance on alumina did not result in
the separation of any significant amount of impurity, a result also given
by the other methods of purification attempted.

The variations shown by the optical rotation of the Type III sub-
stances listed in Table I may perhaps be explained by the fact that in
each instance the insoluble acid was dissolved in a slight excess of
dilute alkali.  It was found that alkaline solutions of the substance
suffered a slow decrease in optical rotation, followed by a slow return
to the original value after acidification.  For instance, in one case
a portion of Preparation 3311, with an initial [cr], of -34.1O showed
a drop in 0.7 per cent sodium hydroxide solution to a value of -21.2',
this returning to -36.0' after acidification.

B. Eydrolytic Products of the Type III Specijc Acid.

As a preliminary step in the study of the products of hydrolysis, the
relation between the specificity, the time of .hydrolysis, the percentage
of reducing sugars, and the optical rotation was studied.

0.0836 gm. of dried Preparation 301, of which the [c& was -3O.!Y, was dfs-
solved in 0.55 cc. of concentrated sulfuric add at -10" and allowed to stand at
this temperature for 1 hour. The viscous solution was then diluted to 21 cc.


740    SPECIFIC SUBSTANCE OF PNEUMOCOCCUS. PAPER III

to give an approximately normal solution of acid, and, before hydrolysis on
boiig, its optical rotation, reducing power, and specificity were tested. The
values observed are to be found in the following table.

tin.                         )M cznl
0        -15.g"           0.0
140        f8.S0          54.8
180       +15.4"          61 .O
300       +22.6'          69.4
360       +23.4"          65.5

Reaction with Type III
antipncu-cur, serum.

++
f

It is thus evident that in boiling normal sulfuric acid the specific
reaction of the Type III acid persists up to the 3rd hour, and that the
maximum reducing power is attained in about 5 hours.

6 gm. of Preparation 301 with a water content of 7.6 per cent, were finely
pulverized and slowly added to a mixture of 30 gm. of concentrated sulfuric acid
and 10 cc. of water at 0'. The acid was stirred slowly with a turbine and after
a short time 10 cc. more of the sulfuric acid mixture were added.  After 2 hours
only a few lumps remained and the mixture was allowed to stand iu the ice box
overnight.  It was then poured into 800 cc. of water and boiled under a reflux
condenser for 5 hours, at which time the reducing power showed a constant
value. The sulfuric acid was removed quantitatively with barium hydroxide.
The barium sulfate was centrifuged off, and the liquid concentrated in uucuo
to 100 cc., boiled with calcium-free Norite, and filtered. The coIorless filtrate
was treated with an excess of basic lead acetate and centrifuged.  The precipi-
tate was washed once with water containing a little basic lead acetate, three
times with alcohol, and was then filtered off, washed with acetone, and dried.
10.7 gm. of the lead salt were recovered in this way.

The supernatant from the basic lead acetate precipitate contained a strongly
reducing substance (0.96 gm. calculated as glucose) and was accordingly treated
with hydrogen sulfide to remove lead, iiltered, and concentrated, finally, in
vacua to dryness in a desiccator. After a few days crystals could be seen with
a hand lens but on account of the diiculty in isolating them in appreciable
quantities the whole was treated with 6 cc. of i:l nitric acid and oxidized as
described previously in this paper.

0.15 gm. of recrystallized potassium acid saccharate was readily obtained.
0.0804 gm. substance gave 0.0282 gm. K&04.
Calculated for COOK(CHOH)~COOH : K, 15.75 per cent.  Found: K, 15.73
per cent.

A similar sugar fraction from the hydrolysis of 4.0 gm. of Type III acid, con-
taining 0.6 gm. of sugar calculated as glucose was de-leaded with hydrogen sul-


M. HEIDELBERGER, W. F. GOEBEL, AND 0. T. AVERY   741

fide and treated in a volume of 50 cc. with 1.3 gm. of phenylhydrazine dissolved
in 3 cc. of 50 per cent acetic acid.  After warming for 1 hour on the water bath
the copious deposit of yellow crystals was Gltered off, washed with 10 cc. of
methyl alcohol to remove tar, tiltered, and dried.  0.40 gm. of osazone was ob-
tained, melting and decomposing at 203-204". A solution of 0.0760 gm. in
7.6 cc. of pyridie-alcohol mixture showed, in a 0.5 dm. tube, a rotation of
- 0.330. After 2 days the reading had decreased to -0.!2', mutarotation in
thii direction being a characteristic of glucosazone (7). Recrystallked for
analysis, the osazone decomposed at 206-210".

Calculated for glucosazone, Ci&fzsO~N~: N, 15.45 per cent. Found: 15.42
per cent.

The salt formed as described above by the addition of basic lead acetate to
the hydrolysis product was suspended in about 300 cc. of water and treated
with hydrogen sulfide.  The lead suhide was removed and the filtrate concentrated
in wcuo to small volume, boiled with Norite, Wered, and the filtrate evapo-
rated to complete dryness over phosphorus pentoxide. 3.1 gm. of a white
glassy mass remained. It was readily soluble in water, diflicultly soluble in
alcohol, and gave a strong glucuronic acid test with naphthoresorcinol. How-
ever, its non-crystalline nature, its insolubility in alcohol, and the di&ulty of
isolating from it a bromophenylosazone did not support its being glucuronic
acid itself.  Moreover its [a& was f12.3' (0.3372 gm. in 20 cc. of water), whereas
the specific rotation of glucuronic acid is +19.1".  It also had a reducing power
of only 48.0 per cent, calculated as glucose. This value could be increased
about 10 per cent by boiling with 0.5 normal hydrochloric acid, while boiling
with hydrobromic acid of the same strength increased the reducing power about
15 per cent and concentrated hydrochloric acid in the cold caused an increase of
only 7 to 8 per cent.  Saccharlc acid could not be isolated from the product
by oxidation with bromine.  The reducing group was also readily oxidized by
nitric acid, but the product again showed remarkable stability toward further
hydrolysis.

It is thus quite certain that the second component of the hydroIysis
products of the Type III acid is not glucuronic acid itself, although
there is evidence that it consists of glucuronic acid combined with
some other substance, perhaps a hexose derivative.  It is hoped to
clear up this point when more material becomes available.

As to the portion of the hydrolysis products not precipitated by
basic lead acetate, however, there can be no doubt that the principal
substance present is glucose, owing to the high yield of glucosazone
obtained, and to the isolation of acid potassium saccharate on oxida-
tion of the sugar.


742    SPECIFIC SUBSTANCE OF PNEUMOCOCCUS. PAPER III

C. Conversion of the Imoluble Type III Acid ido a Soluble Form.

When the insoluble form of the Type III acid, obtained in the usual
way, is boiled with water it gradually enters into solution,

0.1 gm. of Preparation 301 was shaken for 1 hour with 200 cc. of water at
50". Since only a swelling of the granules of the specific substance occurred,
the mixture was diluted to a volume of 500 cc. and boiled under a reflux con-
denser for 14 hours. The resulting clear solution was filtered as a precaution
and concentrated, finally, in a vacuum desiccator until the volume was 5 cc.
The solution, which was still clear, was thrown into 100 cc. of acetone and
filtered. The precipitate was washed with acetone and absolute alcohol and
was dried at room temperature over sulfuric acid at atmospheric pressure until
a constant weight of 0.09 gm. was reached.  The substance thus recovered was
a white amorphous powder soluble in water, but after it was again dried in
wcuo it gradually passed into the insoluble derivative.  l& of the soluble form
was -31.9", and the acid equivalent (352) was in agreement with the values
obtained by solution of the insoluble form in dilute allcali and subsequent
neutralization.

Whether the reversible equilibrium betweenthesoluble and insoluble
forms is one involving simple hydration and dehydration, or one
depending on the opening and closing of a lactone or anhydride group-
ing cannot be decided with the evidence at present available.

DISCUSSION.

The following data are given in order to summarize briefly the
properties of the soluble specific substances of the three fixed types of
Pneumococcus, in the state of purity thus far attained.

The soluble specific substance of Type II pneumococcus is appar-
ently a weakly acidic, nitrogen-free polysaccharide made up chiefly of
glucose units.  Its acid equivalent is about 1250 and the specific opti-
cal rotation is about +74'. It is not precipitated by barium hydrox-
ide or heavy metal salts with the exception of basic lead acetate and
uranyl compounds.  It reacts at a dilution of 1 :S,OOO,OOO with Type
II antipneumococcus serum but does not precipitate Type I and Type
III antisera at a concentration of 1:400.  The substance is converted
by acetic anhydride and pyridine into a very sparingly soluble triacetyl
derivative.

The Type III soluble substance, while also isolated as a nitrogen-


ML. HEIDELBEPGER, W. P. GOEBEL, AND 0. T. AVERY   743

free polysaccharide, is a strong acid with an acid equivalent of about
340, and is made up not only of glucose units but also those of either
glucuronic acid or a derivative of this acid.  It rotates the plane of
polarized light about 33" to the left.  It is precipitated by barium
hydroxide in excess and by heavy metal salts, and is also rendered
insoluble by the addition of strong hydrochloric acid.  In as high a
dilution as 1:6,000,000 it still reacts with Type III antipneumococcus
serum.

The Type I soluble specific substance also appears to be a sugar
derivative, but tiers from the other two substances in its lower
percentage of sugar 1 iberated on hydrolysis and in containing nitrogen
as an apparently essential component.  It rotates the plane of polar-
ized light about 300" to the right, is a strong acid and a weak base, and
is very sparingly soluble at its isoelectric point, which lies at about
pH 4.  In spite of a nitrogen content of 5.0 per cent the substance
gives none of the usual protein color tests.  One-half of the nitrogen
is liberated on treatment with nitrous acid and reducing sugars appear
at the same time, while the specific reaction vanishes.  Under the same
conditions the Type II and Type III substances are untiected by
nitrous acid.  The substance gives the color reaction for glucuronic
acid with naphthoresorcinol, but yields muck acid on oxidation, indi-
cating a relationship to galactose. Since the carbon and hydrogen
contents of the substance are close to the theoretical values for poly-
saccharides it appears possible that in it a nitrogenous sugar deriva-
tive is linked to galacturonic acid through the reducing group of the
latter.  Further evidence on this point will be sought.  The Type I
substance is precipitated by barium hydroxide in excess, by heavy
metal salts, and by phosphotungstic acid.  In the specific precipitin
reaction with homologous Type I antipneumococcus serum it can be
detected in dilutions as great as 1:6,000,000 while at a concentration
of 1:400 it gives a faint cloud with Type III antiserum.

The three polysaccharides contain no sulfur or phosphorus and differ
from the starch-glycogen group of carbohydrates in giving no color
with iodine and in their resistance to the ordinary carbohydrate-
splitting enzymes.

It would be, of course, idle to assume that in their present state of
purity, each of the specific substances represents a defmite chemical


744    SPECIFIC SUBSTANCE OP PNEUMOCOCCUS. PAPER III

compound.  However, iu the case of the three fixed types of Pneu-
~OCOCCUS three totally distinct substances have been isolated from
cultures grown in the same medium.  Successive preparations of the
specific substance have in each instance been quite uniform regardless
of the widely different methods employed in the process of purification.
Moreover, substances reactive to the same degree with homologous
antisera have been derived both from the microorganisms themselves
and from autolyzed broth cultures.

It is thought that these and other considerations based on the data
presented warrant the belief that the three polysaccharides isolated
represent the actual specific substances, stripped of at least a large
portion of accompanying impurities, and that they do not merely
represent inert material carrying an extremely minute amount of the
true specific compounds.

If this be accepted it may be concluded that the soluble specific
substance of each of the three fixed types of Pneumococcus is a dis-
tinct chemical substance, differing in many striking particulars from
the corresponding product elaborated by the other two types, but hav-
ing in common the properties of polysaccharide structure and of resist-
ance to enzyme action.  Each substance breaks down on hydrolysis
into reducing sugars, a part of which, at least, is peculiar to itself.
The Type I substance differs sharply from the other two in containing
nitrogen and in possessing basic as well as acid properties, while of the
other two substances, the Type II is a dextrorotatory weak acid
and the Type III a levorotatory strong acid. Especially striking is
the occurrence of specific substances of such widely differing properties
in microorganisms as closely related as the three fixed types of
Pneumococcus.

The immunological significance of the specific substances has been
discussed by the writers in a recent paper (8) and will therefore not be
touched upon in the present communication.
Many of the questions raised in the course of the work are still under
investigation and the specific substances of other microorganisms are
being studied.

                      SUMMARY.

1. Refinements in the methods for the isolation of the soluble spe-
cific substances of Types II and III pneumococcus are described.


M. ~~ELBERGER, w. F. GOEBEL, AND 0. T. AVJ~RY   745

These improvements have resulted in the isolation of the end-prod-
ucts in a form free from nitrogen and of enhanced activity with
immune serum.

2. The soluble specific substance of Type I pneumococcus is
described and shown to differ sharply from the corresponding sub-
stances of the other two types, each of which, in turn, differs from the
other.

3. Progress is reported on the identification of the sugar units from
which the polysaccharides are built up.
4. The evidence so far accumulated is believed to favor strongly
the view that the polysaccharides isolated are the actual specific
substances of Pneumococcus.

In conclusion the writers wish to express their gratitude to Dr. P.
A. Levene, for his ready counsel and assistance, and to Dr. W. A.
Jacobs for his help as well.

BIBLIOGRAPHY.

1. Heidelberger, M., and Avery, 0. T., J. Exp. Med., 1923, xxxviii, 73; 1924, xl,
  301.

2. Dochez, A. R., and Avery, 0. T., J. ExP. Med., 1917, xxvi, 477.
3. Karrer, P., Scbnider, O., and Smimoff, A. P., Helv. China. Acta, 1924, vii, 1039.
4. Willstitter, R., and Kraut, H., Ber. c&m. Ges., 1923, hi, 149.
5. Felton, L. D., Bosh Med. qnd Surg. I., 1924, cxc, 819.
6. Northrop, J. H., J. Gen. Physiol., 1922-23, 263.
                             v,
7. Levene, P. A., and La Forge, F. B., J. Biol. Gem., 1915, xx, 429.
8. Avery, 0. T., and Heidelberger, M., 1. Exp. Med., 1925, xlii, 367.