220 HAROLD C. ROBINSON AND LEO F. RETTGER 



was biuret-free, and gave a strong ninhydrin test for amino acids. It 

 was subjected to prolonged evaporation on a water bath, with frequent 

 renewal of water. 



Casein product B. 40 grams of crude casein were boiled for eight 

 hours with 200 cc. of 9 per cent HCl under a reflux condenser over a 

 free flame. There was practically no dark residue. The liquid was 

 biuret-free, and gave a strong ninhydrin test. It was heated in an 

 open dish on the water bath until acid vapor ceased to come off. 



Casern product C. 50 grams of the same casein used in B were boiled 

 for eight hours with 200 cc. of 10 per cent HCl over a free flame, with a 

 reflux condenser. The final solution was subjected to distillation, with 

 frequent addition of water. An abundance of fatty-acid-like material 

 came off in the distillate. This product (the distillation liquid residue) 

 was a-biuretic, but gave a strong reaction for amino acids. 



Edestin product. 40 grams of pure re-crystalhzed edestin were heated 

 over a free flame with 10 per cent HCl (reflux condenser) until a biuret 

 test was no longer given. The solution was then heated for several 

 hours in an open dish, to remove as much hydrochloric acid as possible. 



Lactalhumin product. 100 grams of crude commercial milk albumin 

 containing about equal amounts of calcium phosphate and protein 

 were heated two to three hours with 200 cc. of 10 per cent HCl. This 

 treatment brought about a solution of most of the calcium phosphate. 

 The residue of protein was filtered off and heated with 200 cc. of 10 

 per cent HCl until no biuret test could be obtained. The resulting 

 solution was then subjected to prolonged heating in an open vessel, to 

 remove as much as possible of the HCl. Finally the hquid was neu- 

 tralized with NaOH for the precipitation of any remaining calcium 

 phosphate, and filtered. Part of the filtrate was used as such; the 

 remainder was evaporated to a sticky paste. 



These products were all of a very dark brown color, but, like 

 the opsine solution, could be easily decolorized with animal char- 

 coal. For the sake of brevity they will hereafter be designated as 

 "Casein A," "Casein B," "Casein C," "Edestin product" and 

 " Lactalbumin product." The biuret test was negative in all, 

 even by the most delicate methods. As might be expected from 

 the severity of the treatment, all tests for tryptophane were also 

 negative. When, however, a small amount of a weak trypto- 

 phane or peptone solution was added, a positive result was 

 obtained, showing that the negative tests were not due to inter- 



