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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)
Hospital
for Cancer
Research,
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).
Cancer Society
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
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
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
1. Consden,
2. Consden,
R., Gordon,
R., Nature,
A. J. P., Biochem.
J., 38,224
(1944).
TABLE
Distribution
116
3.
4.
5.
6.
7.
8.
ESTIMATION
OF
AMINd
ACID
IN
TISSUE
ARTICLE:
APPLICATION OF PAPER
CHROMATOGRAPHY TO THE
ESTIMATION OF SOME FREE AMINO
ACIDS IN TISSUES OF THE RAT
Jorge Awapara
J. Biol. Chem. 1949, 178:113-116.