Betty Munday and Florence B.

Seibert

TUBERCULIN
IN DETERMINING POLYSACCHARIDE
HAGEDORN-JENSEN METHODS IN
SHAFFER-HARTMANN AND
A COMPARISON OF THE
ARTICLE:
1933, 100:277-285. J. Biol. Chem.

http://www.jbc.org/content/100/1/277.citation
Access the most updated version of this article at

. Sites
JBC Affinity Classics on similar topics on the
Find articles, minireviews, Reflections and
Alerts:

When a correction for this article is posted
When this article is cited
alerts
to choose from all of JBC's e-mail Click here

tml#ref-list-1
http://www.jbc.org/content/100/1/277.citation.full.h
accessed free at
This article cites 0 references, 0 of which can be

b
y

g
u
e
s
t

o
n

O
c
t
o
b
e
r

2
,

2
0
1
4
h
t
t
p
:
/
/
w
w
w
.
j
b
c
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m


b
y

g
u
e
s
t

o
n

O
c
t
o
b
e
r

2
,

2
0
1
4
h
t
t
p
:
/
/
w
w
w
.
j
b
c
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m

A COMPARISON OF THE SHAFFER-HARTMANN AND
HAGEDORN-JENSEN METHODS IN DETERMINING
POLYSACCHARIDE IN TUBERCULIN*
BY BETTY MUNDAY AND FLORENCE B. SEIBERT
(From the Otho S. A. Sprague Memorial Institute and the Department of
Pathology, the University of Chicago, Chicago)
(Received for publication, January 10, 1933)
The following study was undertaken primarily to determine the
best method for and the factors bearing upon the quantitative
determination of the carbohydrate in tuberculin, especially in the
presence of the tuberculin protein. Since the carbohydrate is the
chief admixed substance which is difficult to remove from the pro-
tein during the isolation and purification of the latter, the method
for determining its presence must be accurate and delicate, in
order to detect the smallest traces in the presence of large amounts
of the protein. Furthermore, since the carbohydrate is in the
form of a polysaccharide, a preliminary hydrolysis must be per-
formed before reduction of the copper or other metallic salts is
possible, and because of this preliminary treatment the selection
of the proper method is more than ever important.
Previous reports found in the literature, such as those by Ren-
frew (I), Seibert and Munday (2), and Masucci, McAlpine, and
Glenn (3), all report the use of the Shaffer-Hartmann (4) copper
reagent for quantitatively determining the polysaccharide.
Before the determination a hydrolysis was performed by heating
the preparation for 7 hours with about 3 per cent sulfuric acid.
A pure nitrogen-free polysaccharide isolated by one of the
authors from a human tubercle bacillus culture filtrate made on
Longs synthetic medium, by the second method outlined by
Masucci, McAlpine, and Glenn (3), when analyzed by the method
described above appeared as approximately only 44 per cent
reducing substances, calculated as glucose. This preparation has
* Aided by a grant from the National Tuberculosis Association.
277

b
y

g
u
e
s
t

o
n

O
c
t
o
b
e
r

2
,

2
0
1
4
h
t
t
p
:
/
/
w
w
w
.
j
b
c
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m

Determination of Polysaccharide
been called in this paper tuberculin polysaccharide.
The 44 per
cent obviously does not represent the real amount of polysac-
charide present. The explanation of this fact is that a considerable
portion of the polysaccharide is pentose, as Anderson and Roberts
(5) and Johnson and Renfrew (6) have shown by actual isolation
experiments. Pentose does reduce the Shaffer-Hartmann reagent
but it has low reducing power, a sample of pure arabinose giving
only 45.5 per cent reducing substances, calculated as glucose (see
Table I). The Shaffer-Hartmann method was designed primarily
for determining glucose. Consequently, in a substance such as
the polysaccharide in question, in which there is a mixture of
reducing substances possessing variable reducing powers, there
would be no way of calculating the percentage of each present and
from these results estimating the total amount of carbohydrate.
If the proportionate content of pentose were equal in all tuberculin
polysaccharides, a satisfactory correction might be made, but such
is not the case, as the recent work of Masucci, McAlpine, and
Glenn (7) would tend to show. They found the pentose content of
the polysaccharide made from bovine tubercle bacilli to be practi-
cally negligible compared with that made from the human or
timothy bacillus.
Early in our work, it was noted that the sample of pure nitrogen-
free polysaccharide mentioned above, when hydrolyzed in the same
manner as previously described and then analyzed for its content of
reducing substances by means of the Hagedorn-Jensen (8) ferricya-
nide reduction method, gave 95 per cent1 calculated as glucose.
A similar increase was found when the pure pentose arabinose, and
also the pentosan acacia, was analyzed by this method, as
Table I shows.
It is clear then that substances of low and mixed
reducing powers may be more nearly quantitatively detected by
means of the Hagedorn-Jensen method than by the Shaffer-Hart-
mann method.
On the other hand, the question arises as to whether substances
other than of carbohydrate nature may not also reduce ferricyanide,
since it is apparently so sensitive a reagent.
Especially has one
in mind reducing amino acids which may possibly be released
1 A 5 per cent error was the limit of accuracy obtained with both the
Hagedorn-Jensen and Shaffer-Hartmann methods on the preparations
analyzed in this paper.

b
y

g
u
e
s
t

o
n

O
c
t
o
b
e
r

2
,

2
0
1
4
h
t
t
p
:
/
/
w
w
w
.
j
b
c
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m

B. Munday and F. B. Seibert
279
during the hydrolysis required to split the polysaccharide into its
reducing components. Holden (9) has pointed out that, even
though amino acids do not alone reduce the copper solution, an
error of 10 to 15 per cent may appear due to the oxidation of the
amino acids mixed with glucose.
TABLE I
Comparison of the Two Methods on Different Carbohydrates
Glucose
.......................................
Starch (potato). ..............................
Arabinose .....................................
Gum acacia
...................................
Tuberculin polysaccharide. ....................
TABLE II
Per cent reducing substances,
calculated as glucose
Sh&3f-
HartIIlaIlIl
microcuprous
method
.
93.0
87.7
45.5
47.2
43.9
Hagedorn-
Jensen method
93.0
86.4
94.4
78.6
95.3
Comparison of the Two Methods on Three Amino Acids
Amino acid
Tryptophane
No treatment. . . .
HotH.S04.................................
Phenylalanine
No treatment. . . . .
Hot HzSOa.................................
Proline
No treatment. . .
Hot H&Sod. . . . . .
I
Per cent reducing substances,
calculated a8 glucose
22
20
0
3
0
3
J
-_
-
Hagedorn-
lensen method
59
69
0
10
0.9
19.0
Pure tryptophane, phenylalanine, and proline were studied with
this idea in mind and tryptophane alone showed considerable
reduction; i.e., 22 per cent, calculated as glucose, with the Shaffer-
Hartmann reagent and 59 per cent with the Hagedorn-Jensen
reagent. When, however, the amino acids were subjected to a
preliminary treatment for 7 hours with hot sulfuric acid in exactly

b
y

g
u
e
s
t

o
n

O
c
t
o
b
e
r

2
,

2
0
1
4
h
t
t
p
:
/
/
w
w
w
.
j
b
c
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m

280
Determination of Polysaccharide
the same manner as the polysaccharide-containing substances
would be treated, the tryptophane showed very little more reduc-
tion than before the treatment, but the phenylalanine and proline
now showed an increase in reducing powers, especially when
determined by the Hagedorn-Jensen method (see Table II).
Obviously, then, it is necessary to make the quantitative deter-
mination of reducing substances in the absence of free amino acids
and reducing substances other than the carbohydrate itself.
This
end result. may theoretically be accomplished in either of two ways:
first, by .removing the protein before hydrolysis, or secondly, by
hydrolyzing first and then precipitating out of the hydrolysate the
nitrogenous hydrolyt,ic products. Both methods were studied.
Four precipitants for removing nitrogenous subst.ances either
before or after hydrolysis were used as follows:
1. The Folin-Wu (10) reagents were added to the protein solu-
tion; as recommended by Shaffer and Hartmann (4), 5 cc. of 10
per cent sodium tungstate and 5 cc. of $ N HzS04 were added in a
final volume of 100 cc., and the resulting precipitate was filtered off.
2. The Somogyi (11) method of adding 8 volumes of Reagent I
(12.5 gm. of ZnSOl . 7Hz0 dissolved in water f 125 cc. of 0.25 N
H2S04 and made to 1 liter) and 1 volume of Reagent II (0.75 N
NaOH). This mixt,ure was well sbken and the precipitate re-
moved by filtering through a dry paper.
Or the method of pre-
cipitating with ZnSOa, recommended by Hagedorn and Jensen (8),
was used in some cases.
3. Mercuric nitrate precipitation was carried out exactly as
described by Shaffer and Hartmann (4) for use with urine, except
that HzS was used instead of Na2S to remove the last traces of
mercury. The mercuric nitrate solution was prepared according
to Patein and Dufau (12). Following this precipitation, the
Hagedorn-Jensen method could not be used, since the zinc present
in the reagents formed nitrites from the nitrates, thus interfering
with the iodometric titration.
4. Mercuric sulfate precipitation (13).
A 10 per cent solution
of mercuric sulfate was prepared by dissolving 73 gm. of red
mercuric oxide in 1 liter of 4 N HzS04.
Equal volumes of this solu-
tion and the unknown were mixed and the precipitate was filtered
off. An aliquot of the filtrate was treated with an excess of water-
washed H,S to remove the mercury, and the excess of H:S in

b
y

g
u
e
s
t

o
n

O
c
t
o
b
e
r

2
,

2
0
1
4
h
t
t
p
:
/
/
w
w
w
.
j
b
c
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m

B. Munday and F. B. Seibert
281
turn was removed by aeration. The sample was made up to a
definite volume and filtered and the filtrate brought just to
neutrality with solid sodium bicarbonate.
Precipitation of Nitrogenous Xubstances before Hydrolysis- In
this type of experiment, the solid material to be analyzed, 40 to
160 mg. according to the amount of polysaccharide estimated to be
present, was put into a small amount of solution. To it were added
the various precipitants described above, so that the final filtrate
consisted of about 25 cc. of solution. Of this, 15.6 cc. + 0.5 cc. of
concentrated H&304 were hydrolyzed in a boiling water bath for 7
hours. The solution was then neutralized with NaOH and made
to 25 cc. The Shaffer-Hartmann microcuprous titration deter-
minatior? was carried out upon 5 cc. samples of this solution and
the Hagedorn-Jensen determination on approximately 1 cc.
samples.
Table III contains the pertinent results obtained by these pro-
cedures.
The results in Table III show several facts. In the first place,
the Folin-Wu reagent does not remove the tryptophane, thus
allowing it to appear in the filtrate and consequently to give
erroneous results. This reagent is, therefore, not a satisfactory
precipitant for mixtures containing free amino acids.
Since the zinc sulfate precipitation did not remove tryptophane
even as effectively as did the Folin-Wu reagent, there was no
advantage in using this method of precipitation.
The two mercury precipitation methods were effective in remov-
ing tryptophane, within a 1 per cent error, but they had the dis-
advantage also of removing polysaccharide and could not, there-
fore, be used for precipitating preliminary to a hydrolysis, in a
process aiming to determine the amount of polysaccharide present.
2 The reagent as used was made as follows: 25 gm. of Na&03 (anhy-
drous), 20 gm. of NaHC03, 25 gm. of Rochelle salt, 7.5 gm. of CuSOd, and 100
cc. of 0.1 N solution of KIO, per liter. 5 cc. of this solution + 5 cc. of un-
known in a large Pyrex test-tube were covered with a glass bulb and heated
10 minutes in a vigorously boiling water bath, cooled, and then 1 cc. of 2.5
per cent KI, 5 cc. of N HxS04, and 1 cc. of 1 per cent starch solution were
added. The mixture was then titrated with 0.005 N Na&O, and the titra-
tion difference between this value obtained and the value resulting from a
similar blank determination was divided by 8.5 to give directly in mg. the
glucose present in 5 cc. of solution.

b
y

g
u
e
s
t

o
n

O
c
t
o
b
e
r

2
,

2
0
1
4
h
t
t
p
:
/
/
w
w
w
.
j
b
c
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m

Determination of Polysaccharide
Of these procedures, therefore, the most satisfactory is a pre-
liminary precipitation with the Folin-Wu reagent followed by a
hydrolysis. In this way the protein is completely removed while
at the same time the entire amount of polysaccharide is allowed to
pass into the filtrate where the reducing content is determined,
preferably by the Hagedorn-Jensen method. Masucci, McAlpine,
and Glenns (3) contention that reducing substances may be
TABLE III
Removal of Nitrogenous Substances before Hydrolysis
/ j Per cent reducing substances, calculated as glucose
Substance hydrolyzed
following precipitation
1 t$ regnt j ;;&T j ZnSOa 1 HgSOa
Tryptophane.. 20
Phenylalanine . .
Proline . . .
j 3
/ 3
Tuberculin polysac- I
charide. . . . . 0 / 44
Tuberculin protein1
I
fraction.. . 4.6 30
I

$
9.01 11


$
15.2/ 1
H.J.&.-H. H.-i
I.S.-H.IH.-J.lS.-H./H.-J
~_
I
_I- -
69 1st 56t 24 68 0.5 1.1
10
/
19
i 6 13
2 7
31
H&NO&
S.-H. /H.-J.
1
* S.-H. means Shaffer-Hartmann method; H.-J. means Hagedorn-Jensen
method.
t These two figures wTere obtained on tryptophane that had been pre-
cipitated with the Folin-Wu reagent but the filtrate then was not hydro-
lyzed, in contrast to all other determinations listed in the table.
$ These fractions represent stages in the purification of the protein and
were chosen because of the varying quantities of polysaccharide admixed
with the protein.
removed by this tungstate reagent, if the precipitation is per-
formed first, is not borne out by the present investigation.
Precipitation of Nitrogenous Substances after Hydrolysis--In
this type of procedure, the 12 to 100 mg. of material in 15.6 CC. of
solution were mixed with 0.5 cc. of concentrated sulfuric acid and
hydrolyzed for 7 hours in a boiling water bath.
The hydrolysate
was neutralized, made up to 25 cc., and then precipitated by means
of an equal volume of mercuric nitrate or mercuric sulfate solution,
as described above, the precipitate discarded, and the filtrate

b
y

g
u
e
s
t

o
n

O
c
t
o
b
e
r

2
,

2
0
1
4
h
t
t
p
:
/
/
w
w
w
.
j
b
c
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m

B. Munday and F. B. Seibert 283
tested for reducing substances by the Shaffer-Hartmann and
Hagedorn-Jensen methods. The Folin-Wu precipitation method
was not used in these cases following hydrolysis, since this reagent
will not remove amino acids. Table IV shows the results obtained.
The data in Table IV show that the precipitation with the mer-
cury reagents after hydrolysis does not materially interfere with
the accuracy of the determination of the reducing content of the
polysaccharide, whether the polysaccharide was alone or present
with protein. In other words, as can be seen by comparing Table
IV with Table III, similar results were obtained, within experi-
mental error, whether the hydrolysis was performed before mer-
curic nitrate or mercuric sulfate precipitation or whether it was
TABLE IV
Removal of Nitrogenous Substances after H&-o&is
Substance hydrolyzed before
precipit&ion
0
S.-H.* H.-J.* S.-H. H.-J. S.-H. H.-J.
-- -- --
Tuberculin polysaccharide 44 95 43 90 42
Tuberculin protein frac-
tion.. . . . . . . . . . . . 30 57 28 63 25
I
11 26 9 24 7
I
1, 10 1 2 0.6
* S.-H. means Shaffer-Hartmann method; H.-J. means Hagedorn-
Jensen method.
4.6
9.0
15.2
Per
cent N
Per cent reducing substances, calculated as glucose
No reagent HgSO4
performed after a precipitation with the Folin-Wu reagent. The
latter method is, however, simpler and should be used whenever
one is certain that free ammo acids and other reducing substances
are not present in considerable quantities, as is the case with the
protein fractions analyzed in this paper. Everett and Sheppards
finding (14) that mercuric salts and alkali do not completely pre-
cipitate all the nitrogenous substances from biological fluids, must
be considered in this connection, although from the data above it
would seem that such non-precipitable compounds are not released
in significant amounts during the hydrolysis of the tuberculin
protein.
This type of investigation is therefore necessary before bacterial
polysaccharides, as well as other complex polysaccharides con-

b
y

g
u
e
s
t

o
n

O
c
t
o
b
e
r

2
,

2
0
1
4
h
t
t
p
:
/
/
w
w
w
.
j
b
c
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m

284 Determination of Polysaccharide
taming carbohydrate components other than glucose, can be
determined quantitatively.
SUMMARY
The Hagedorn-Jensen ferricyanide method detects reducing
substances of lower reducing power than glucose (pentose, amino
acids, etc.) and is therefore more satisfadtory when one is analyz-
ing the tuberculin polysaccharide, which contains a considerable
portion of pentose, than is the Shaffer-Hartmann microcuprous
titration method.
The polysaccharide when alone or in combination with tuber-
culin protein is not precipitated by the Folin-Wu tungstate rea-
gent, but is removed to a considerable extent by mercuric nitrate
or mercuric sulfate.
The amino acid tryptophane is almost completely removed by
mercuric nitrate or mercuric sulfate, but not by the Folin-Wu
tungstate reagent or by zinc sulfate.
The most satisfactory procedures for quantitatively determin-
ing the tuberculin polysaccharide are, therefore, as follows: (1)
If the polysaccharide is alone or in combination with whole pro-
tein, as it usually exists in tuberculin, and if free amino acids or
other reducing electrolytes are not present in significant amounts,
a procedure consisting of precipitation by means of the Folin-Wu
reagent, followed by a hydrolysis for 7 hours with 3 per cent
sulfuric acid, neutralization, and then a determination of the
reducing power by the Hagedorn-Jensen ferricyanide reagent,
yields accurate results, within about a 5 per cent error.
(2) If
free amino acids and other nitrogenous electrolytes are present,
a hydrolysis followed by the mercuric sulfate precipitation is ad-
visable. The reducing substances are then determined in the
filtrate by means of the Hagedorn-Jensen ferricyanide reagent.
BIBLIOGRAPHY
1. Renfrew, A. G., J. Biol. Chem., 83,569 (1929).
2. Seibert, F. B., and Munday, B., Am. Rev. Tuberc., 23, 23 (1931).
3. Masucci, P., McAlpine, I<. L., and Glenn, J. T., Am. Rev. Tuberc., 22,
669 (1930).
4. Shaffer, P. $., and Hartmann, A. F., J. Biol. Chem., 46, 365 (1920-21).
5. Anderson, R. J., and Roberts, E. G., Am. Rev. Tuberc., 22, 664 (1930);
J. Am. Chem. Xoc., 62, 5023 (1930).

b
y

g
u
e
s
t

o
n

O
c
t
o
b
e
r

2
,

2
0
1
4
h
t
t
p
:
/
/
w
w
w
.
j
b
c
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m

B. Munday and F. B. Seibert
6. Johnson, T. B., and Renfrew, A., Am. Rev. Tuber-c., 22, 655 (1930).
7. Masucci, P., MeAlpine, K. L., and Glenn, J. T., Am. Rev. Tuberc.,
24,737 (1931).
8. Hagedorn, H. C., and Jensen, B. N., Biochem. Z., 136,46 (1923).
9. Holden, H. F., Biochem. J., 20, 263 (1926).
10. Folin, O., and Wu, H., J. Biol. Chem., 38, 81 (1919).
11. Somogyi, M., Proc. Sot. Exp. Biol. and Med., 26, 353 (1929).
12. Patein, G., and Dufau, E., J. pharm. et chim., 16, 221 (1902).
13. West, E. S., Scharles, F. H., and Peterson, V. L., J. Biol. Chem., 82,
137 (1929).
14. Everett, M. R., and Sheppard, F., Proc. Am. Sot. Biol. Chem., 8, p.
lxxxi (1932); J. Biol. Chem., 97, p. lxxxi (1932).

b
y

g
u
e
s
t

o
n

O
c
t
o
b
e
r

2
,

2
0
1
4
h
t
t
p
:
/
/
w
w
w
.
j
b
c
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m