A CRITICAL EVALUATION OF HAHN’S QUANTITATIVE

METHOD FOR DETERMINING PROTEIN

AND PROTEOSE.”
BY FLORENCE B. SEIBERT.
(From the Department oj Pathology of the Cniversity of Chicago and the
Otho S. A. Sprague Memorial Institute, Chicago.)

(Received for publication, July 23, 1926.)

An accurately standardized method for determining quantita-

tively the content of whole protein and of proteose in a solution
is much to be desired. Especially is this true when attempts are
made, as with tuberculin, to ascertain whether the biological
activity of a preparation is associated with the protein or with the
smaller proteose molecules derived therefrom.
Such a method will, of necessity, employ as reagents, protein
precipitants. Many studies have been made by previous in-
vestigators to select the precipitant which will differentiate most
sharply between the whole protein molecule and its smaller frag-
ments-the proteoses. Trichloroacetic acid is generally accepted
to be the best reagent for this purpose. This acid was used in
1892 by Frankel (1) to free glycogen of protein. Starling (2)
found that it would not precipitate Griibler’s commercial “pep-
tone” but that it was efficient in separating the coagulable pro-
teins in plasma, serum, and blood. Greenwald (3) employed
trichloroacetic acid as a precipitant in the estimation of non-
protein nitrogen in blood, and showed that when it was followed
by kaolin treatment, the protein was precipitated completely.
Amino acids are not adsorbed by the precipitate.
Hiller and Van Slyke (4) found that whole protein from blood is
completely precipitated by trichloroacetic acid and that the pre-
cipitate does not contain free monoamino acids or peptide nitro-
gen. If it is desirable to precipitate, along with the protein, all
* Aided by a grant to Dr. Esmond R. Long from the Medical Research
Committee of the National Tuberculosis Association.
265

This is an Open Access article under the CC BY license.

266 Protein and Proteose Determination

of the decomposition products except amino acids, they recom-

mend picric and tungstic acids as most efficient, and meta-phos-
phoric acid, colloidal iron, and mercuric chloride, as less efficient.
A quantitative comparison of the efficiency of phosphomolybdic,
meta-phosphoric, and trichloroacetic acids, as precipitants, was
made by Sjollema and Hellerschy (5). Their results led them
to conclude that trichloroacetic acid precipitated only the whole
protein and that phosphomolybdic acid and meta-phosphoric
acid precipitated a part of the decomposition products along with
the protein.
With this information Hahn (6) designed a method for deter-
mining quantitatively the amount of protein decomposition
products in the serum of patients suffering from different diseases.
He precipitated the serum with 2.5 per cent trichloroacetic acid
and determined the nitrogen in the filtrate. A similar amount of
serum was precipitated with phosphotungstic acid and again the
nitrogen determined in this filtrate. The difference between these
two nitrogen figures represents the nitrogen in the protein decom-
position products or proteose fraction. He determined the effi-
ciency of the method by showing that casein which had been
hydrolyzed for a short time gave a precipitate with phospho-
tungstic acid but none with trichloroacetic acid, and also that uric
acid and creatinine were not precipitated by phosphotungstic
acid. It is this method which has been critically examined in this
investigation.
Method.
A 1 per cent protein suspension was prepared and duplicate 1
cc. samples were used for total nitrogen determinations. The
solution was filtered through hardened filter paper and again 1
cc. samples were tested for total nitrogen; this figure being the
soluble total nitrogen.
Two 2 cc. samples were precipitated with 8 cc. of 20 per cent
trichloroacetic acid, making a 16 per cent concentration, and
after standing 15 minutes the precipitate was filtered off through
hardened filter paper. The filtrate was always clear. 1 cc. of
concentrated HGS04 was added to 8 cc. of the filtrate in a large
Pyrex test-tube, and the liquid evaporated over the free flame until
it charred. Nitrogen was determined by the micro-Kjeldahl
method, as outlined below.
 F. B. Seibert 267
Two more 2 cc. samples were precipitated with 8 cc. of 5 per
cent phosphotungstic acid (containing 30 cc. of concentrated H&04
per liter), and this precipitate was filtered off through hardened
paper and 8 cc. of the filtrate evaporated with 1 cc. of concen-
trated HzS04 to the fuming stage and then digested as usual.
This digestion requires constant attention. The mixture bumps
badly because of the large amount of solid inorganic matter which
is unavoidably present.
The nitrogen in the filtrate from 1 cc. of protein solution pre-
cipitated by phosphotungstic acid is the residual nitrogen, or the
nitrogen of the monoamino acids and undetermined nitrogen.
The nitrogen in the filtrate from 1 cc. of protein solution precipi-
tated by trichloroacetic acid is the proteose plus residual nitrogen,
and therefore, by difference between these two filtrate nitrogens,
the proteose nitrogen is obtained. (This proteose nitrogen figure
really includes diamino acids, since they have been shown by Van
Slyke (7) to be precipitated by phosphotungstic acid. The error
is so small, as illustrated below, that it can be disregarded in this
work.) The difference between the nitrogen in the trichloroacetic
acid filtrate and the total nitrogen is equal to the whole protein
nitrogen. Blanks were subtracted in all cases.
A very important factor for consideration in this method is the
concentration of trichloroacetic acid to be used. Hiller and
Van Slyke (4) showed that the percentage of nitrogen in the
filtrate from a trichloroacetic precipitation of Witte peptone
varied with the amount of trichloroacetic acid used; for example,
after precipitation with 2.5 per cent there was 85.4 per cent N in
the filtrate; with 5 per cent, 77.9 per cent N; with 10 per cent,
62.5 per cent N. With the sample of Witte peptone used by the
author, 16 per cent trichloroacetic acid was used and no more
precipitate was produced by the addition of more acid. Only
52.2 per cent of the original nitrogen was found in the filtrate.
It had been found on different occasions that 10 per cent acid is
not sufficient to produce complete precipitation and so a minimum
of 16 per cent was used as a routine and in all of the tests made
this was found to be adequate. A 16 per cent concentration of
trichloroacetic acid does not hydrolyze proteins under the condi-
tions of these experiments. This is amply illustrated in the sub-
sequent pages and can be inferred from the results of Greenwald,
268 Protein and Proteose Determination
who was able to show that a 22 per cent concentration was not
hydrolytic.
In determining residual nitrogen, tungstic acid is recommended
by Hiller and Van Slyke and should theoretically be a better
precipitant than phosphotungstic acid, since it will not precipitate
the diamino acids. The bumping which occurs with this reagent,
however, is so much worse than when phosphotungstic acid is
used, that a comparison of the determination with the two reagents
was made. The residual nitrogen in crystalline ovalbumin, found
by means of phosphotungstic acid, was 0.1 per cent N; by the
tungstic acid, 0.085 per cent N. In two casein preparations the
residual nitrogen determined by means of phosphotungstic acid
precipitation was 0.18 per cent N and 0.14 per cent N; by tungstic
acid, 0.18 per cent N and 0.15 per cent N respectively. The two
methods checked so closely that it was deemed justifiable to use the
former in the following analyses.
The nitrogen in the filtrates was determined by the micro-
Kjeldahl method. The accuracy of the results was not affected
by the presence of the trichloroacetic and phosphotungstic acid
reagents, and, therefore, no attempt was made to remove them
before continuing with the analyses. After the filtrates were
concentrated with HzS04 to the fuming stage, solid Na2S04
(about 1 gm.) and a small piece of CuSOb were added and they
were digested for + hour after they were colorless. After cooling,
about 5 cc. of distilled water were added. 6 cc. of a saturated
solution of NaOH were introduced through a pipette, after the
distillation apparatus (Koch’s modification of Folin’s apparatus)
was closed. The ammonia was distilled over into ~/lo0 HCl and
the distillate titrated with alizarin and ~/lo0 NaOH. Duplicate
samples were run in all cases and no results were accepted which
varied more than 0.2 cc. ~/lo0 alkali, making an error of about 1
per cent or less.
Results.
Determinations of the percentage of whole protein, proteose,
and residual nitrogen were made on a variety of highly purified
proteins and when possible upon crystallized proteins. In order
to determine the applicability of the method to proteins in general,
representatives of four different protein groups were chosen;
 F. B. Seibert 269
crystallized egg albumin (albumin), crystallized edestin (globulin),
gliadin (prolamin), two samples of casein (phosphoprotein), and
Witte peptone and a proteose fraction of the latter. Table I
shows that all of the whole proteins, which had been purified by
the best methods known at the present time, are precipitated
quantitatively (within at most 2 per cent) by trichloroacetic acid,
under the conditions specified. From these data, the conclusions
can therefore be drawn that trichloroacetic acid precipitates pure
proteins practically quantitatively, and this supports Greenwald’s
findings for the proteins of the blood.

TABLE I.

Substance tested.
T Total W.

A5 protein. is proteose. ls residual.

_-
per cent per cent per cent
Albumin.
Crystalline Ovslbumin 1. . .. .. ... ... . 99.19 0.00 0.08
“ “ 2. .. .. .. .. ... . 98.32 0.83 0.83
Impure Ovalbumin 1. . . . . .. . .. . 86.15 13.13 0.71
“ “ 2. . .. . ... .. . . 68.80 16.89 14.30
Globulin.
Edestin, crystalline. ... ... .. .. .. .. . 98.73 0.63 0.63
Prolamin.
Gliadin (Vickery) . .. ... ... . . .. .. ... 97.80 0.73 1.47
Phosphoprotein.
Casein (author). . .. .. .. ... .. .. ... 98.61 0.00 1.46
“ (Osborne) .. .. . . .. . . .. . .. 98.24 0.68 1.06
Witte peptone. . . ... .. . . . ... . ... .. 47.63 44.55 7.81
Proteose . . . . .. . .. ... .. . ... .. 0.00 75.28 24.79
-

On the other hand, by this method, impure proteins are shown

not to contain 100 per cent whole protein. Two samples of egg
protein, for example, which correspond to the fraction precipitated
by the addition of more (NHI)$04 after the crystalline oval-
bumin is removed (or chiefly, the conalbumin fraction and some
proteose) are seen to contain comparatively less whole protein
(GS.80 per cent and 86.15 per cent) and much more proteose (16.89
per cent and 13.13 per cent) than the crystalline protein itself.
And as might be expected, even more proteose (44.55 per cent)
is found in Witte peptone.
270 Protein and Proteose Determination
If we can assume, therefore, that crystalline proteins and pro-
teins prepared by repeated solution and reprecipitation and dialysis
consist of only whole protein molecules, and if trichloroacetic
acid precipitates them practically quantitatively while it will
precipitate only the whole protein from admittedly impure protein
fractions or from Witte peptone, then trichloroacetic acid can
be used as a reagent for separating whole protein from proteose.
The one further question to be answered before the absolute
accuracy of such a method can be determined, is whether this
reagent will be as efficient in precipitating whole protein when
proteose is also present. An experiment was therefore designed
to test this point. Proteose was purified by discarding from Witte

TABLE II.

Total N.
Total N.
As protein. A3 proteose. As residual.

per cent per cent per cent per cent

Crystalline ovalbumin. ....... 13.18 98.33 0.83 0.83
Proteose..................... 13.31 0.00 75.28 24.79
Mixture of above. ........... 13.24 52.79 34.82 12.38
“ (by calculation). .... 13.24 48.94 38.21 12.83
Difference. ................. 0.00 +3.85 -3.39 -0.45

peptone all that would precipitate with trichloroacetic acid,

dialyzing the clear filtrate containing only the proteose and residual
nitrogen and drying the non-dialyzable residue. Nitrogen parti-
tion analyses were run upon (1) the crystallized ovalbumin alone,
(2) on the proteose alone, and (3) on a mixture of equal parts
ovalbumin and proteose. The results are contained in Table II.
A comparison of the results obtained on the mixture, with the
theoretical, show that when the protein and proteose were mixed,
3.85 per cent more nitrogen was found in the whole protein frac-
tion than there should be and on the other hand, 3.39 per cent
lessof the nitrogen as proteose and 0.45 per cent lessof the nitrogen
as residual was found in the mixture. This is interpreted to
mean that in the concentrations used, 3.39 per cent of the proteose
and 0.45 per cent of the residual nitrogen were carried down from
solutions of mixtures of protein and proteose with the trichloro-
 F. B. Seibert 271

acetic acid precipitate and were then erroneously considered as

whole protein. So that the method here outlined is accurate with-
in about 3.5 to 4.0 per cent.
Furthermore, attention should be called to the fact that this
degree of accuracy will be obtained only when the concentration of
protein solution here recommended (namely, 0.01 gm. protein
per cc.) is used. Determinations were run upon the same protein
(impure egg albumin) when it existed in concentrations of 0.1 gm.
per cc. and 0.01 gm. per cc. There appeared to be 4.5 per cent
more whole protein nitrogen and 3 per cent more proteose nitrogen
precipitated from the more concentrated solution than from the
dilute one, and a corresponding loss in the residual nitrogen.
The precipitate in the more concentrated solution was very thick
and presumably occluded considerable residual nitrogen, and
proteose nitrogen.
Concentration
of impure
egg albumin. Protein N. Proteose N. Residual N.
gm. per CL. per cent per cent per cent
0.01 68.8 16.9 14.3
0.1 73.3 19.9 6.8

SUMMARY.

Hahn’s method, with modifications as described, is reliable

with an experimental error of no more than 1 per cent for deter-
mining the percentage of whole protein, proteose, and residual
nitrogen.
Highly purified and when possible crystalline representatives of
different protein groups were quantitatively precipitated, to
within 1 to 2 per cent, by trichloroacetic acid. This finding sup-
ports the conclusions of Greenwald and others with blood proteins.
Impure ovalbumin preparations were shown to contain only 86.15
per cent and 68.8 per cent and a sample of Witte peptone only
47.63 per cent whole protein nitrogen by this method.
When equal parts of a purified protein and a pure proteose are
mixed, trichloroacetic acid precipitates the whole protein quanti-
tatively, but in addition, carries down with the precipitate some
of the proteose and residual nitrogen, which then is erroneously
considered as whole protein. Within this limit the method is
accurate. In the experiment here described 3.85 per cent of the
272 Protein and Proteose Determination

proteose and residual nitrogen was included in the whole protein

fraction.
A considerable error is introduced when a protein solution ten
times as concentrated as that recommended (1 per cent) is used,
because of occlusion of the decomposition products with the whole
protein precipitate.

Sincere appreciation is expressed to Dr. Milton T. Hanke for

his many helpful suggestions and criticisms.

BIBLIOGRAPHY.

1. FrBnkel, S., Arch. ges. Physzol., 1892, lii, 125.

2. Starling, E. H., J. Physiol., 1893, xiv, 131.
3. Greenwald, I., J. Biol. Chem., 1915, xxi, 61.
4. Hiller, A., and Van Slyke, D. D., J. Biol Chem., 1922, liii, 253.
5 Sjollema, B., and Hellerschy, C. W. G., Biochem. Z., 1917, lxxxiv, 371.
6. Hahn, A., Biochem. Z., 1921, cxxi, 262.
7. Van Slyke, D. D., J. Biol. Chem., 1915, xxii, 281.