j. biol. chem.-1933-munday-277-85

8/11/2019 J. Biol. Chem.-1933-Munday-277-85

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Betty Munday and Florence B. SeibertTUBERCULIN

INDETERMINING POLYSACCHARIDE

HAGEDORN-JENSEN METHODS IN

SHAFFER-HARTMANN AND

A COMPARISON OF THE

ARTICLE:

1933, 100:277-285.J. Biol. Chem.

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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

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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 al l 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

Haged orn-Jensen and Shaffer-Hartmann metho ds on the preparations

analyzed in this paper.

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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

...................................

Tub ercu lin polysacch aride. ....................

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 Acid s

Amino acid

Tryptophane

No treatment. . . .

HotH.S04.................................

Phenylalanine

No treatment. . . . .

Hot HzSOa.................................

Proline

No treatment. . .

Hot H&S od. . . . . .

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

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280


the same manner as the polysaccharide-containing substances

would be treated, the tryptophane showed very lit tle 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

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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 Roch elle 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 gla ss 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

glucos e present in 5 cc. of solution.

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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 I I I

Removal

of Nitrogenous Substan ces 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

Tube rculin 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 The se two figures wTere obtained on tryptophane that had been pre-

cipita ted with the Folin-Wu reagent but the filtrate then was not hydro-

lyzed, in contrast to all other determinations listed in the table.

Thes e fractions represent stages in the purification of the protein and

were chosen becau se of the varying quan tities 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

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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-

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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. Bio l. Chem ., 83,56 9 (1929).

2. Seibert, F. B., and Munday, B., Am. Rev. Tuberc., 23, 23 (1931).

3. Masucc i, P., McAlpine, I


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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. Bio l. 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).

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