APPLICATION OF PAPER CHROMATOGRAPHY TO THE ESTIMATION OF SOME FREE AMINO ACIDS

IN TISSUES OF THE RAT*

BY JORGE AWAFARA

(From The University of Texas, M. D. Anderson Hospital for Cancer Research, Houston)

(Received for publication, October 21, 1948)

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.

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 min- utes. 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. Spectro- photometric readings were compared with readings obtained for known solutions of amino acids treated in a similar manner.

* This work was supported in part by a grant from the American Cancer Society (grant No. INSTR 23).

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114 ESTIMATION OF AMINO ACID IN TISSUE

EXPERIMENTAL

Fig. 1 indicates that the four fractions found in rat liver fall within the range of aspartic acid, glutamic acid, glycine, and alanine. Other amino acids were present in lower concentrations and a quantitative estimate was impossible. It can be observed that aspartic acid and glutamic acid are free of interference from any of the amino acids under study. Glycine 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.

FIG. 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. Glu- tamine, which moves at the same rate as alanine, in the chromatogram, is converted into pyrrolidonecarboxylic acid during the extraction procedure. The latter compound does not interfere, as it does not appear in the chromat- ogram.

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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Glycine

Amino acid nitrogen per gm. fresh tissue Animal No.

Aspartic acid Glutamic acid

Y Y

1 50 57 2 47 77 3 68 64 4 43 62 5 61 50 6 50 56 7 72 92 8 48 45 9 46 60

10 54 54

7 Y

80 86 loo 120 150 85 120 74 85 68 80 78

110 70 100 60

90 82 95 I 70

J. AWAPARA 115

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 amino acid distribution in liver. It has also been found of great value in demonstrating the conversion of histidine to glutamic acid by the action of rat liver extracts. Table I shows the relative concentration of aspartic acid, glutamic acid, glycine, and alanine in normal rat liver. Extension of

TABLE I

Distribution of Amino Acid Nitrogen in Some Fractions of Liver

Average. . . 53.9 1 61.7 101.0 I 79.3

this procedure to kidney, spleen, heart, and skeletal muscle indicates that the same four amino acids exist in these tissues in similar concen- trations.

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 spectro- photometric analysis of the colored solution produced by ninhydrin.

BIBLIOGRAPHY

1. Consden, R., Gordon, A. H., and Martin, A. J. P., Biochem. J., 38,224 (1944). 2. Consden, R., Nature, 162, 359 (1948).

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116 ESTIMATION OF AMINd ACID IN TISSUE

3. Naftalin, A., Nature, 161, 763 (1948). 4. Woiwod, A. J., Nature, 161,169 (1948). 5. Fisher, R. B., Parsons, D. G., and Morrison, G. A., Nature, 161, 764 (1948). 6. Awapara, J., Arch. Biochem., 19, 172 (1948). 7. Williams, R. J., and Kirby, H., Science, 107, 481 (1948). 8. Consden, R., Gordon, A. H., and Martin, A. J. P., Biochem. J.,-42,443 (1948)

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Jorge Awapara

ACIDS IN TISSUES OF THE RATESTIMATION OF SOME FREE AMINOCHROMATOGRAPHY TO THE APPLICATION OF PAPERARTICLE:

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

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