IX] PROTEINS AND AMINO- ACIDS 155 



A separation of pepsin from erepsin has been achieved in the case 

 of the seeds of the Hemp (Cannabis sativa) by means of the different 

 solubilities of the two enzymes in water and salt solutions. 



Expt. 145. ^The extraction and the separation of the two enzymes^ erepsin and 

 pepsin^ from Hemp-seed (Cannabis sativa) ^ Weigh out 50 gms. of hemp-seed, grind 

 it in a coffee-mill and extract with 250 c.c. of 10 % sodium chloride solution. Allow 

 the mixture to stand all night and then filter. Both operations should be carried 

 out at as low a temperature as possible. Measure the filtrate, and add acetic acid 

 to the extent of 0*2 %. A dense precipitate is formed. Filter again, keeping as cool 

 as possible. 



The acid filtrate contains the erepsin, but not the pepsin. Measure out 40 c.c. 

 into each of three small flasks, and add the following : (i) 0*2 gm. of Witte's peptone, 

 (ii) the same, only boil the whole solution, (iii) 0*2 gm. of carmine fibrin 2. Add a 

 little toluol to all three flasks, plug with cotton-wool, and incubate for three to four 

 days. Test for tryptophane in flasks (i) and (ii) ; the first gives a marked reaction, 

 the second little or no reaction. The fibrin in (iii) will remain unaltered. 



The precipitate produced by the acetic acid is then washed on the filter twice 

 with 100 c.c. of 10 % sodium chloride solution, containing 0*2 % acetic acid, to 

 remove traces of erepsin. The precipitate is then treated with about 70 c.c. of 

 water, allowed to stand for a time, and then filtered. The filtrate is divided into 

 three equal portions. Add the following respectively : (i) O'l gm. of carmine fibrin, 

 (ii) the same, but the solution is boiled, (iii) 0'2 gm. of Witte's peptone. Add a little 

 toluol to all three flasks, plug with cotton-wool and incubate for 3-4 days. The 

 fibrin will be seen to digest slowly in flask (i) : (ii) will show no digestion, and 

 (iii) will give no tryptophane reaction. 



REFERENCES 



Books 



1. Abderhalden, B. Biochemisches Handlexikon, iv. Berlin, 1911. 



2. Osborne, T. B. The Vegetable Proteins. London, 1909. 



Papers 



3. Blood, A. F. The Erepsin of the Cabbage [Brassica oleracea). J. Biolog, 

 Chem., 1910-1911, Vol. 8, pp. 215-225. 



4. Chibnall, A. C, and Schryver, S. B. Investigations on the Nitrogenous 

 Metabolism of the Higher Plants. Part I. The Isolation of Proteins from Leaves. 

 Biochem. J., 1921, Vol. 15, pp. 60-75. 



5. Dean, A. L. On Proteolytic Enzymes. I. Bot. Gaz., 1905, Vol. 39, 

 pp. 321-339. 



1 Vines, S. H. Ann. Bot., 1908, Vol. 22, pp. 103-113. 



2 Freshly washed and finely chopped fibrin is placed in carmine solution (1 gm. carmine, 

 1 c.c. of ammonia, 400 c.c. of water) for 24 hrs. Then strain off and wash in running 

 water till washings are colourless. 



