2058 E. ROBERTS AND D. G. SIMONSEN 
during acid hydrolysis of the tissue proteins. Hydrolysis of the protein-free extracts 
at all stages of autolysis in the various tissues revealed little liberation of peptidic 
material. 
From the above discussion and results presented, it appears highly unlikely that 
the amino acids and other ninhydrin-reactive constituents which are found in 
extracts of fresh tissues arise by autolytic and proteolytic breakdown of the proteins 
after excision of the tissue. The results indicated that the changes in these constit- 
uents probably occur relatively slowly enough so that precautions such as freezing 
of tissue prior to dissection of specimens does not appear to be necessary. 
CHANGES IN THE CONTENT OF EASILY EXTRACTABLE NINHYDRIN-REACTIVE 
CONSTITUENTS DURING DEVELOPMENT 
Although each tissue of a mature organism of a particular species has a characteristic 
distribution of free or easily extractable amino acids and related substances, changes 
in the content of these constituents per unit of fresh weight may take place at all 
stages of development until the final adult functional and structural patterns are 
laid down. Tissues from the frog, chick embryo (including yolk, albumen and mem- 
branes), salamander, mouse and other organisms have been analyzed for their 
content of free amino acids at different stages of development and compared with 
adult tissues. 
It was found that ovarian eggs of Rana pipiens contained higher concentrations 
of free amino acids than shed unfertilized eggs from which the jelly coat had been 
removed. After fertilization there was an increase in the content of free glycine at the 
two-cell stage, while taurine was first detected in the late gastrula state. In other 
stages of the development of Amblystoma punctatum the pattern of free amino 
acids of the yolk was different from that of the neural plate and ectoderm. The free 
amino acid pattern of the blastodisc of the unincubated fertilized chicken egg was 
very similar to that of the yolk. However, after the 1st day of incubation the blasto- 
disc, made up of adhering yolk and embryonic tissue, was found to contain taurine, 
an amino acid which was not detected in the yolk at any stage. In the case of the 
chick embryo, alcoholic extracts made from 150mg of fresh weight of albumen 
showed only traces of freely extractable ninhydrin-reactive material, possibly pep- 
tidic. Only at 16 days of incubation did small amounts of some free amino acids 
appear (Fig. 5g) and remain at similar concentrations to 20 days. In Fig. 60 is shown 
a chromatogram from the hydrolysate of the mixed proteins of the albumen. The 
finding of a remarkably small pool of free amino acids at the time that rapid utiliza- 
tion of the albumen is taking place by the embryo suggests that the albumen pro- 
teins may be transported out of the albumen as intact units, or at least as relatively 
large polypeptide units, for use elsewhere in the embryo. The changing pattern in 
the yolk as a function of time of incubation of the chick embryo is shown in Figs. 
61-64. As in the case of the albumen, at no time did the free amino acids reflect the 
amino acid composition of the mixed yolk proteins. 
In Figs. 65-68 are shown the amino acid patterns in the liver of the tadpole and 
adult bullfrog. Particularly noteworthy was the progressive increase in the taurine 
content with the stage of development, although other changes, both increases and 
decreases, in the content of various constituents were noted as well. Examination of 
References p. 348/349 
