138 H. K. MITCHELL AND J. R. SIMMONS 
GENERAL EXTRACTION METHOD 
In preparing the extracts already described conditions of —20° and +90° were used 
and this is essential to avoid enzyme action as shown in Fig. 1. Neither cold-alcohol 
extracts nor hot-water extracts show such changes though the former do contain 
substances unstable to heat alone. For these reasons we have retained the use of low- 
temperature grinding and extraction and have developed a general method that 
appears applicable to any kind of tissue (Fig. 3). Furthermore, it provides a sharp 
preliminary fractionation as will be shown (Figs. 4 and 5). 
A 10-g sample of go-h larvae (from egg laying) was washed thoroughly with distilled 
water and blotted dry with filter paper. The live animals were added quickly to a 
pre-cooled solution of 30 ml of methanol and 10 ml of water contained in the steel 
cup of an Omnimixer (Serval). This in turn had been pre-cooled in a dry ice—cellosolve 
mixture and this was maintained during 10 min grinding at maximum speed. The 
BS 

ML 
p MOLES / 
N 
% 40 80 120 160 200 
FRACTION NO. 
Fig. 2. Column fractionation of an extract of Drosophila larvae. Extract: cold aqueous propanol 
followed by hot-water extraction of 60 g (wet wt.) of middle third instar larvae. Column: water 
jacketed (22°) Dowex-50-4X column 2.2 x 45cm. Equilibrated with starting buffer from acid 
form and washed with distilled water. Elution: gradient elution with a 1r00-ml mixing vessel was 
used starting with ammonium formate (pH 2.49), 0.05 M with respect to NH,* in both mixing 
vessel and solvent reservoir. The gradient was started after collection of 75, 15-ml fractions with 
the sequence as follows: 

240 

Molarity Change in 
Buffer pH (NH,*) reservoir at 
fraction No. 
Ammonium formate 2.9 O.1 75 
Ammonium formate 353 0.15 142 
Ammonium formate 3.65 0.2 191 
Ammonium acetate 5-5 0.4 259 
Ammonium acetate 6.8 0.6 294 
Ammonium acetate 8.0 1.0 359 
References p. 146 
