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APPLICATION

OF PAPER
CHROMATOGRAPHY
ESTIMATION
OF SOME FREE AMINO
IN TISSUES OF THE RAT*
BY JORGE
(From

The

University

of Texas,

M.

TO THE
ACIDS

AWAFARA

D. Anderson
Houston)

(Received for publication,

Hospital

for Cancer

Research,

October 21, 1948)

Methods
Tissue extracts were prepared free of proteins and lipides, as recently
described (6). Chromatographic analyses of extracts were carried out
in accordance with the modification of Williams and Kirby (7). For
quantitative estimation of amino acids, 0.1 ml. of extract was used, divided
into five approximately equal small spots applied at 2 cm. intervals on
Whatman filter paper No. 4. Chromatography was carried out for 18
hours with redistilled phenol saturated with water. Amino acids were
located on the chromatogram with a 0.05 per cent ninhydrin solution in
butanol. The spots developed on the chromatogram were cut out of the
paper, placed in test-tubes, and 2 ml. of a 1 per cent ninhydrin solution
added. The addition of 1 ml. of a 10 per cent aqueous pyridine solution
was found favorable. The tubes were placed on a water bath for 20 minutes. Full color development was obtained at the end of this period. The
colored solution was transferred to a 25 ml. volumetric flask, made to
volume, and read in the Beckman spectrophotometer at 570 rnp. Spectrophotometric readings were compared with readings obtained for known
solutions of amino acids treated in a similar manner.
* This work was supported
(grant No. INSTR 23).

in part by a grant from the American


113

Cancer Society

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The development of paper chromatography by Consden, Gordon, and


Martin (1) has opened a new approach to the problem of resolving and
detecting small amounts of substances in a complex biological mixture.
Although this technique was originally designed for the qualitative analysis
of protein hydrolysates, a number of publications have appeared in which
numerous adaptations and improvements are described (2) as well as
attempts to use paper chromatography quantitatively
(3-5).
The present work was undertaken to ascertain the applicability of this
procedure to the study of amino acid metabolism.

114

ESTIMATION

OF

AMINO

ACID

IN

TISSUE

EXPERIMENTAL

Fig. 1 indicates that the four fractions found in rat liver fall within the
Other amino
range of aspartic acid, glutamic acid, glycine, and alanine.
acids were present in lower concentrations
and a quantitative
estimate
was impossible.
It can be observed that aspartic acid and glutamic acid
Glycine
are free of interference from any of the amino acids under study.
is shown to be well separated from threonine and to overlap slightly taurine
and serine. Below the alanine spots are shown ten amino acids which do
not interfere with any of the four fractions found in rat liver extracts.

1. Paper chromatogram

of known amino acid solutions

and rat liver extract

Possible interference from peptides, glutamine, and asparagine was


taken into consideration. The presence of peptides in any of the four
fractions was ruled out by elution of the individual fractions followed by
acid hydrolysis (8). No changes were observed upon chromatography of
the hydrolysates. Asparagine was shown to have the same RF value as
glycine. The presence of asparagine was ruled out by the failure to
demonstrate aspartic acid upon hydrolysis of the glycine fraction.
Glutamine, which moves at the same rate as alanine, in the chromatogram, is
converted into pyrrolidonecarboxylic acid during the extraction procedure.
The latter compound doesnot interfere, asit doesnot appear in the chromatogram.
The reproducibility of the method was shown by analysis of an alanine
solution containing 100 y of amino nitrogen per ml. The results of twenty

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FIG.

J.

115

AWAPARA

separate determinations
showed an average of 103 y of amino nitrogen,
with a coefficient of variation of 5 per cent.
Paper chromatography,
in its present state, offers numerous possibilities
as a quantitative
method in the study of amino acid metabolism.
In this
laboratory it has been used in studying the effect of adrenalectomy
on the
It has also been found of great value in
amino acid distribution
in liver.
demonstrating
the conversion of histidine to glutamic acid by the action of
rat liver extracts.
Table I shows the relative concentration
of aspartic
Extension
of
acid, glutamic acid, glycine, and alanine in normal rat liver.

of Amino

Acid
Amino

Animal

in

acid nitrogen

Some

Fractions

of Liver

per gm. fresh tissue

No.
Aspartic

acid

Glutamic

50

47
68
43
61
50
72
48
46
54
. .

acid

Glycine

1
2
3
4
5
6
7
8
9
10
Average.

Nitrogen

53.9

57
77
64
62
50
56
92
45
60
54

loo
150
120
85
80
110
100
90
95

61.7

101.0

80

86
120
85
74
68
78
70
60
82
70
79.3

this procedure to kidney, spleen, heart, and skeletal muscle indicates


that the same four amino acids exist in these tissues in similar concentrations.
SUMMARY

Paper partition chromatography


has been applied to the estimation of
some amino acids present in rat liver extracts.
The presence of glutamic
acid, aspartic acid, glycine, and alanine has been demonstrated.
A
quantitative
estimation of these fractions was made possible by spectrophotometric
analysis of the colored solution produced by ninhydrin.
BIBLIOGRAPHY

1. Consden,
2. Consden,

R., Gordon,
R., Nature,

A. H., and Martin,


162, 359 (1948).

A. J. P., Biochem.

J., 38,224

(1944).

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TABLE

Distribution

116
3.
4.
5.
6.
7.
8.

ESTIMATION

OF

AMINd

ACID

IN

TISSUE

Naftalin, A., Nature, 161, 763 (1948).


Woiwod, A. J., Nature, 161,169 (1948).
Fisher, R. B., Parsons, D. G., and Morrison, G. A., Nature, 161, 764 (1948).
Awapara, J., Arch. Biochem., 19, 172 (1948).
Williams, R. J., and Kirby, H., Science, 107, 481 (1948).
Consden, R., Gordon, A. H., and Martin, A. J. P., Biochem. J.,-42,443 (1948)

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ARTICLE:
APPLICATION OF PAPER
CHROMATOGRAPHY TO THE
ESTIMATION OF SOME FREE AMINO
ACIDS IN TISSUES OF THE RAT

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Jorge Awapara
J. Biol. Chem. 1949, 178:113-116.

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