M. J. Blish 509 
from this source is introduced in the case of flour extracts, not- 
withstanding the fact that under certain conditions copper is 
known to be eapable of forming insoluble compounds with a 
few amino-acids, though not with the majority of them. 
When water extracts of flour were allowed to. stand for several 
weeks at room temperature in the presence of toluene, and ana- 
lyzed at intervals for non-protein nitrogen by the copper method, 
a gradual increase in non-protein nitrogen by autolysis occurred 
although there was no sign of putrefaction. The amount of 
total non-protein nitrogen doubled during the period from May 
25 to July 6, 1917. This indicates that the method is appli- 
cable to proteolysis studies in flour.® 
SUMMARY. 
1. Practically a complete separation of protein from non- 
protein nitrogenous substances in water extracts of wheat flour 
may be accomplished by treating the extract with 0.1N NaOH 
followed by 0.1 Nn CuSO, until there is but slightly more CuSO, 
than an exactly equivalent amount of NaOH. The method is 
simple of manipulation and leaves no troublesome excess of the 
reagents employed for the precipitation. The method permits 
of rapid filtration through ordinary filter paper, giving a water- 
clear solution which may be readily concentrated to one-twen- 
tieth its original volume, for determinations of amino nitrogen by 
Van Slyke’s micro method, and for amide nitrogen determinations. 
Some peptide nitrogen is not precipitated by the copper method, 
but the removal of the true proteins is practically complete. 
2. Normal patent flour contains but about 2 mg. of amino-acid 
nitrogen for every 100 gm. of flour, and about three times as 
much nitrogen in free acid amide form. 
3. There is probably a considerable amount of non-protein ni- 
trogen not precipitated by the copper method which is neither 
amino-acid nitrogen nor is it in the form of peptide complexes; 
its nature is not known. 
4. The method is applicable to studies of proteolysis or other 
studies involving the estimation of protein cleavage products in 
wheat flour. 
5. It is not unlikely that the method will be found applicable 
to biological extracts from other sources than wheat and flour. 
6 Acknowledgment is made to Miss Erma Lessel, who performed a large 
part of the preliminary analytical work. 
