IMMUNOCHEMICAL STUDIES OF THE PLANT PROTEINS 419 
Method of Preparation 
Proteins Soluble in Aqueous Salt Solution 
Since these proteins occur mainly in the embryo of the wheat 
grain and not very much in the endosperm (Osborne and Campbell, 
'00), ordinary white bread or pastry flour should not be used. The 
commercial "entire wheat" preparations give very good results except 
that the yield is small. 
Leucosin, being soluble in pure water, can be extracted from the 
flour by simply soaking in cold water (with the addition of some pre- 
servative as thymol or toluol to prevent bacterial decomposition) for 
a few days, and decanting the supernatant fluid which then contains 
this protein together with some globulin, dissolved by virtue of the 
mineral salts contained in the grain, and proteose, together with 
sugars, etc. This method of preparing leucosin was discarded early 
in the work because it was found much easier to extract it together 
with the globulin and separate them afterwards. So the entire wheat 
flour was stirred in lo percent salt solution (about 1,200 gm. to 3,500 
cc.) and allowed to stand at room temperature for about three days 
(no preservative is necessary). The flour settles to the bottom and 
the supernatant solution (which is pinkish and syrupy) can be siphoned 
off. It is desirable to allow the flour to separate completely from the 
supernatant fluid so that further clearing will be unnecessary, for the 
viscosity of this solution renders it difficult to filter. Less than one 
half of the volume of the salt solution is recovered, the rest remaining 
entangled in the flour, so it is profitable to make a second extraction 
from the same flour by adding a volume of salt solution equal to that 
removed by decantation. This second extraction is almost as rich 
in protein as the first. Since the globulin is insoluble in water at 
neutral reaction it may now be separated out by dialyzing the whole 
solution in water until free from CI (the other proteins may be separ- 
ated from each other as will be subsequently shown), or all the proteins 
may be salted out together by saturation of the extract with am- 
monium sulphate, and separation effected by dialysis after again 
being dissolved. 
In the preparation of natural proteose it was found desirable to 
follow the former m.ethod. When the salt extract is freed from NaCl 
by dialysis all of the globulin and possibly parts of some of the other 
proteins are thrown out of solution and can be removed by filtration. 
