LOUIS FREEDMAN 27 



fullers earth, was subjected to hydrolysis by adding concen- 

 trated sulphuric acid until a 30% solution was obtained and 

 boiling for 36 hours. This hydrolysate failed to give any growth 

 stimulating activity on either streptococci or yeast. 



It is therefore apparent that most or all of the vitamine was 

 removed from the casein as described. This shows that the 

 active substance in the protein is not necessarily a constituent 

 part of the protein molecule, and does not have to be isolated by 

 breaking up the protein.* 



SUMMARY OF CONCLUSIONS. 



1. We can obtain from purified casein, commercial gelatin, 

 yeast protein and edestin, by hydrolysis of these proteins, cer- 

 tain substances which show marked growth stimulating activity 

 on hemolytic streptococci. Hydrolysates of purified egg globulin, 

 lactalbumin and hordein, show traces, whereas hydrolysates of 

 the other proteins examined are devoid of these activating sub- 

 stances. 



2. We have found that these active substances are not a 

 constituent part of the protein molecule, and that the amount 

 of the substances present in the protein depends on the physical 

 and adsorptive properties of the protein and the method and de- 

 gree of purification. 



3. The properties of these active substances, as obtained 

 from the proteins described above, show that they are probably 

 related to if not identical with the water-soluble vitamines ob- 

 tained from brewers' yeast, particularly vitamine D. 



4. We have described a method for determining the amount 

 of these active substances by the use of Sorensen's indicator 

 method, which is based on the change in hydrogen ion concentra- 

 tion due to the fermentative action of the bacteria. This method 

 is advocated for comparative results only, and not as a direct 

 quantitative method. 



* The experiments with neutral sodium caseinate, just described, may be of practical 

 importance in that they may lead to a method of preparing a vitamine-free casein. The 

 casein thus obtained gives positive tests for all the color reactions known for the various 

 mino acids, while its nitrogen content is the same as that of the original casein from which 

 it was prepared. Further work is now in progress on this problem. 



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