952 Non-Protein Nitrogen of Wheat Flour 
removed by filtration. This principle in modified forms has 
. since been made use of by different investigators, although the 
accuracy of the separation of protein from non-protein nitrogen 
by this method has always been questioned. 
Osborne and Leavenworth? have recently reported a study of 
copper-protein compounds, using edestin and gliadin in their ex- 
periments, and have found that if the correct amount of copper 
sulfate is added to a solution of gliadin in dilute sodium hydroxide 
solution, the gliadin and copper are both ‘practically completely 
precipitated. As they state, however, there are several points 
to be definitely cleared up if this procedure is to be made a basis 
for the accurate separation of protein from non-protein nitrogen 
compounds in extracts from. biological material. 
When the principle of Osborne’s procedure was tried with flour 
extracts in this laboratory, it was found that more nitrogen was 
removed by this means than by the use of any other of the re- 
agents previously mentioned. Phosphotungstic acid removed 
nearly, but not quite, as much and tannic acid slightly less. More- 
over, the copper-protein precipitate filtered readily, giving a 
water-clear solution, which could easily be concentrated under 
reduced pressure to one-twentieth of its original volume. That 
there was probably no copper in the filtrate, other than that which 
was in combination with amino-acids and peptides, was indicated 
by the fact that no blue color was perceptible, nor were positive 
tests with potassium ferrocyanide obtained until after consider- 
able concentration. ‘The method seemed so simple of manipula- 
tion that it was decided to test the effectiveness of the separation 
by ascertaining whether or not all protein was removed, as well 
as to test the copper-protein precipitate for free amino nitrogen 
as an indication of the presence or absence of less complex nitrogen 
compounds in the copper-protein precipitate. 
In Table I are data which indicate the total nitrogen which is 
not precipitated from a water extract of a standard patent flour 
(Ceretana) by the copper method, as compared with the total 
nitrogen not removed from the same extract by tannic acid, col- 
loidal iron, and phosphotungstic acid, respectively. The figures 
represent averages of several determinations by each method. 
2 Osborne, T. B., and Leavenworth, C. S., J. Biol. Chem.., 1916-17, 
Xxviil, 109. 
