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In an earlier report from this laboratory (10) the changes in protein nitrogen and various forms of nonprotein nitrogen-amino, amide, peptide, ammonia, and urea-were reported for the different tissues of peas and pea seedlings during the early stages of growth. The data emphasize the quantitative importance of the free amino acids, particularly in the roots and shoots of these young seedlings. Although they contribute only about 3 % to the total nitrogen of the seed, their contribution rises to 10 % in the 5-day seedling. In root shaft and shoot shaft tissue of the 5-day seedling, from 40 to 45 % of the nitrogen is in the free amino acids. The work did not make clear the role these amino acids plav in the conversion of the storage proteins of the seed to the functional proteins of the young plant. Clearly the first information needed for this purpose is a dcetailed picture of the changes in the concentration of the individual amino acids. For the former method, the extracts were evaporated to dryness under vacuum, and taken up either in water followed by adjustment to about pH 2 with a few drops of HCl, or in citrate buffer of pH 2.35 (11). At this stage, the cotyledon extracts were filtered and made to a convenient volume for the ion exchange chromatography. The other extracts, however, gave better chromatographic separations, if they were first extracted with petroleum ether before filtration and dilution to a convenient volume.The method of Rosen (13) was used with minor modifications to determine the ninhydrin color yield of the fractions from the column. The concentration of the ninhydrin reagent was increased to 5 %, since the 2 ml fractions from the fraction collector resulted in increased dilution of the reaction mixture. Twice the recommended volume of the 50 % isopropanol diluent was used. We found the cyanide-acetate solution not to be stable (7) in that a lower ninhydrin color was obtained if the solution used was more than 4 hours old. Hence the cyanide and acetate components were mixed just before use. The fractions, except for the less acidic ones from the 15 cm column for the basic amino acids, were partially neutralized with 2 drops of 2 N NaOH before treatment with the reagents. Standard leucine samples were run at regular intervals to check the performance of the method.For the automatic method, the extracts were evaporated to dryness under vacuum, and simply dissolved up to a convenient volume in pH 2.91 citrate buffer (12). In general, the results by the 2 methods agreed well, with the exceptions mentioned below, and were averaged together for the results presented in table I. Amino acids were determined on hydrolysates of some of the residues from the 70 % alcohol extractions. The residues containing 8 to 25 mg of nitrogen were hydrolzed by autoclaving them at 121°for 5 hours with 25 ml of 3 N HCl. The amino acid contents of aliquot portions were determined using the manual (fraction collector) method.

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