AMINO ACIDS AND DERIVATIVES IN DROSOPHILA 139 
resulting slurry was poured into a 60-ml medium-porosity glass funnel, pre-cooled at 
—20° ina deep freezer. Cold 50% methanol was added in batches to a total of 200 ml. 
The resulting filtrate (Fraction 1, Figs. 3 and 4) was evaporated to a syrup in a rotary 
vacuum evaporator at 35°. The residue was washed with 30 ml of methanol (100%), 
50 mlof ether or chloroform and again with ro ml of methanol, all at — 20° with gravity 
filtration. The residue was quickly sucked dry with a water pump and the combined 
filtrates evaporated in vacuo to give Fraction 2. The dry residue was added to 30 ml 
OMNIMIXER 
2 parts METHANOL 
Frazen T/SSUE 
2 parts 50% METHANOL 



m DEEPFREEZER 
-20° 
Oa | METHANOL 
followed by 
SO%METHANOL., | | 5 parts ETHER 

below 35° 
AMINO ACIDS 
Evaporated in vacuo 
SMALL PEPTIDES 
Evaporated in vacuo 
LIPID DERIVATIVES 
/ 
RESIQUE into 3ports 
of water above 90° 
FL EROGCOTIELGC ee = oo >3 
3 extractions 
lLyophilize 
| 
4 
LARGE PEPTIDES 
| 
RESIVE §=——————_—_——>4_ PROTEIN 
Fig. 3. A diagram showing details of tissue extraction procedure. 
of water in an Omnimixer cup contained in water above go°. After a 3-min mixing 
(in the hot-water bath) the material was filtered with suction through the original 
glass funnel. The extraction was repeated twice more and the combined filtrate was 
lyophilized to give Fraction 3. The residue in the funnel was then extracted twice 
with 0.1 M NaOH by gravity filtration at less than 4°. Protein was precipitated by 
10%, trichloroacetic acid, washed with trichloroacetic acid and dried or redissolved, 
as desired, to yield Fraction 4. The method is diagrammed in Fig. 3. 
An example of results obtained from an application of the extraction method as 
detailed above is summarized in Fig. 4. The data were obtained by photometric 
analyses of paper chromatograms (see analytical methods). Several points are worthy 
of note. First, before hydrolysis, Fractions 2 and 4 are negligible and Fraction 1 
contains three-quarters of the ninhydrin-reacting material. By hydrolysis this fraction 
increases less than 2-fold indicating a principal content of free amino acids and smaller, 
more soluble derivatives. On the other hand Fraction 3 increases nearly 1o-fold on 
References p. 146 
