1 1 1 
Vines . — The Proteases of Plants ( V ). 
large quantity of water, will yield an active solution. It is the fibrin- 
digesting extract that offers difficulties. 
Whilst experimenting with Hemp-seed, it struck me that this would be 
suitable material for the attempt to isolate the proteases. It is unnecessary 
to give all the tentative efforts that were made. It will suffice to say that 
I was guided by the fact with which I had long been familiar, and which is 
especially brought out by the experiments described in the first part of the 
present paper, that the fibrin-digesting protease is more soluble in NaCl- 
solutions than in water. Moreover, I was aware that NaCl-solutions also 
extract a great deal of proteid from seeds ; it therefore seemed to be 
probable that the precipitation of the proteid in such an extract would 
carry down with it the fibrin-digesting protease. This probability I have 
succeeded in realizing. I found it to be necessary to use a strong (io°/ o ) 
NaCl-solution, and to obtain a strong extract holding much proteid in solution. 
On acidifying such an extract with the least possible quantity of acetic 
acid, a dense precipitate of proteid was formed. Filtering this off, the acid 
filtrate was found to peptolyze actively, and to have no action on fibrin ; 
the fibrin-digesting protease evidently remained in the precipitate on the 
filter. Washing the precipitate with io°/ 0 NaCl-solution, slightly acidulated 
with acetic acid, the washings were found at first to act upon Witte-peptone, 
but this action gradually diminished and eventually ceased. A portion of the 
washed precipitate was then extracted with distilled water and filtered ; the 
somewhat opalescent filtrate was found to digest fibrin actively, and not to 
act upon Witte-peptone, as indicated by the absence of the tryptophane- 
reaction. Further details are given in the following account of typical 
experiments : — 
50 grms. crushed Hemp-seed were extracted with 250 cc. 10% NaCl-solution, 
and left to filter all night at a low temperature. It may be remarked that, as 
filtration is slow, it is necessary that the temperature should be as low as possible 
during the process. The filtrate was a rather viscid liquid, giving dense precipitate 
on boiling, also a strong tryptophane-reaction, and digesting fibrin actively. To this 
filtrate acetic acid was now added to 0-2%, and a dense precipitate was produced; 
the liquid was then put to filter in the cold. 
The acid filtrate gave turbidity on boiling, and marked tryptophane-reaction : 
its digestive properties were tested as follows : — 40 cc. were placed in each of three 
bottles: to No. 1, no proteid was added; to No. 2, o-i grm. fibrin; to No. 3, 0-2 grm. 
Witte-peptone. After 24 hours’ digestion in the incubator the tryptophane-reactions 
were: — No. 1, marked; No. 2, marked; No. 3, strong; the fibrin in No. 2 was un- 
altered. The experiment with No. 2 was continued for three days longer, at the end of 
which time the fibrin still remained unaltered. Thus the acid filtrate digested Witte- 
peptone but not fibrin. 
The precipitate produced by acetic acid was washed on the filter with 100 cc. 
10% NaCl-solution containing 0-2% acetic acid, and the digestive activity of the 
