THE CLEAVAGE OF PROTEINS WITH ALKALI 117 



present interfered with the reaction, but by adding the 

 solution all or nearly all at once it was possible to obtain 

 comparative results. Experiments with the haptophor of 

 the colon bacillus had shown that the maximum reduction 

 was obtained by boiling for two and one-half hours with 

 2.5 per cent, hydrochloric acid (see p. 70). 



Three grams of the haptophor of egg-white was mixed 

 with 200 c.c. of water, and 20 c.c. of 25 per cent, hydro- 

 chloric acid. A second sample was prepared in the same 

 way except that it was filtered before adding the acid. 

 Both were boiled with reflux condenser. After boiling half 

 an hour and then at intervals of three hours, aliquot parts 

 were removed, neutralized, titrated with Fehling's solution, 

 and the amount of reducing substance calculated. Other 

 samples were hydrolyzed with sulphuric acid, with less 

 satisfactory results. These preliminary experiments indi- 

 cated that the reducing substance is all present in the 

 portion soluble in water, and that the maximum yield, 

 which if calculated as dextrose, is about 9 per cent., is 

 obtained by boiling from ten to twelve hours, and until the 

 mixture no longer gives the biuret test. 



Accordingly, 25 grams of the egg-white haptophor was 

 shaken for two hours on a shaker with ten times its weight 

 of water, filtered, 200 c.c. more of water added, the solution 

 neutralized with hydrochloric acid, then 50 c.c. of 25 per 

 cent, hydrochloric acid added, thus making approximately 

 a 5 per cent, solution of material in 2.5 per cent. acid. This 

 was boiled with a reflux condenser for ten or twelve hours, 

 until the solution no longer gave the biuret test. It was 

 then filtered, leaving very little on the filter. The clear, 

 red-brown filtrate was cooled, neutralized with sodium 

 hydroxide, and benzolated by the Schotten-Baumann 

 method. The mixture became very warm, but was cooled 

 by surrounding the flasks with pounded ice and salt. When 

 the reaction ceased, the compound settled nicely, and was 

 filtered by suction after standing two or three hours. The 

 precipitate was washed with water containing a little 

 ammonia, and treated with boiling water, in which a large 



