140 THE PROTEINS AND PROTEASES [CH. 



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 O2 %. 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) O2 gm. of Witte's 

 peptone, (ii) the same, only boil the whole solution, (iii) 0'2gm. of carmine fibrin 1 . 

 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) 0*1 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. Biocheuiisches Handlexikon, iv. Berlin, 1911. 



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



PAPERS 



3. Blood, A. P. The Erepsin of the Cabbage (Brassica oleracea). J. Biolog. 

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



4. Chittenden, R. H., and Mendel, L. B. On the Proteolysis of Crystallized 

 Globulin. J. Physiol., 1894, Vol. 17, pp. 48-80. 



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

 pp. 321-339. 



6. Dean, A. L. On Proteolytic Enzymes. II. Bot. Gaz., 1905, Vol. 40, 

 pp. 121-134. 



7. Osborne, T. B. Proteids of the Flax-seed. Amer. Chem. J., 1892, Vol. 14, 

 pp. 629-661. 



8. Osborne, T. B. Crystallised Vegetable Proteids. Amer. Chem. J., 1892, 

 Vol. 14, pp. 662-689. 



9. Osborne, T. B. The Proteids of Barley. J. Amer. Chem. Soc., 1895, Vol. 17, 

 pp. 539-567. 



1 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. 



