I40 H. K. MITCHELL AND J. R. SIMMONS 






Ninhydrin before Ninhydrin after 
hydrolysis hydrolysis 
Fraction == — SES 
Dry weight Dry weight 
pemole|g % total pmole |g % total 
T. 50% methanol 210 75 360 17 
2. CHCl,—methanol —_ ~- 10 0.5 
3. Water (100°) 69 25 637 31 
4. Protein-TCA — — 1080 52 

Fig. 4. Distribution of amino acids, peptides and other derivatives among the fractions obtained 
by: (1) 50% methanol at — 20°; (2) methanol-ether; (3) hot water; (4) cold alkali. The data are 
for third instar larvae but those from first instar larvae and embryos are similar. 
hydrolysis and its total amino acid content is nearly double that of Fraction 1. With 
respect to the total balance of amino acids (Fig. 4, last column) it may be observed 
that about half the amino acids in the larvae are present as protein and, the other 
half, the pool from which protein could be made, is present largely in the form of 
peptides and amino acid derivatives. 
ANALYTICAL METHODS 
In nearly all of the work reported here amino acids and peptides were determined by 
paper chromatography followed by a photometric determination of the color pro- 
duced with ninhydrin. Although this method is not as precise as others that could be 
used it has the advantage of giving additional information besides amounts. That is, 
in analyzing successive fractions from a column one can see at a glance when changes 
in composition occur both in unhydrolyzed and hydrolyzed fractions. 
In detail the most commonly used procedure was as follows: Aliquots of fractions 
(3-5 wl) containing 0.02-0.1 wM of ninhydrin-reacting material were pipetted onto 
sheets (20 * 23 cm) of washed filter paper (Whatman No. 1, washed by descending 
chromatography for 36 h with 3°% aqueous NH) with hydrolyzed and unhydrolyzed 
samples adjacent. Hydrolysis was carried out using 50 wl of sample and 0.1 ml of 
6 N HCl in a sealed tube for 16h at 105°. After hydrolysis the acid was removed 
im vacuo over KOH and P,O,. After redissolving in water an appropriate aliquot was 
spotted beside the unhydrolyzed sample. Each sheet for chromatography was also 
spotted with 3 or 5 wl of an equal mixture of known amino acids (glutamic acid, 
glycine, alanine, valine and leucine) at a total concentration of 0.02 M. Chromato- 
grams were then irrigated, four or five at a time in battery jars, by ascending chromato- 
graphy with -propanol: 1% aqueous NH, (2: 1). After 8 h the sheets were dried 
in air and dipped into 1°% ninhydrin in acetone. Usually color was allowed to develop 
in the dark at 35° for 12-16 h. Sheets were then cut in appropriate strips and total 
color was measured using a Spinco Analytrol. In our experience only proline and hy- 
droxyproline gave seriously low values (less than 50%). Cystine was usually approx. 
20°% low but most of the other amino acids gave color values within 10% of the 
maximum (represented by glutamic acid or valine). Peptides occasionally gave 
peculiar colors and tended to give low values for one terminal amino acid’. In spite 
References p. 146 
