CHEMICAL CONSTITUENTS 291 



From this altered SSS, four samples were prepared by various alka- 

 line treatments and tested both chemically and biologically in compari- 

 son with the original material. Each of the samples may be considered 

 separately. The original material (A) gives tests for aldehyde groups, 

 is high in "acetyl" content, and is optically as well as immunologically 

 active. In B, a portion of A treated for 30 minutes in N/10 NaOH at 

 100°C, the optical activity is lost, the aldehyde groups are removed, 

 "acetyl" content and all immunological activities are greatly reduced. 

 In C, sample B treated with NH 4 OH, there is a reduction of acid from 

 vacuum distillation, no indication of sugar on acid hydrolysis, but an 

 increase of the iodine reaction. Optical rotation is changed from zero to 

 -}-190 o , or higher than the control. Immunologically also this sample is 

 more active than the original material (A). Sample D is the original 

 material (A) treated with NH 4 OH. There is a decrease of 90% in acid 

 on vacuum distillation, a slight decrease in glucose number, a higher 

 optical rotation than the original material, as well as a high titer in all 

 immunological tests. It is significant that with a 90% decrease in "ace- 

 tyl" content the immunizing activity is higher than the original. There 

 is also a significant increase in its combining equivalent with antibody. 

 Sample E is the NH 4 OH treated control (D) further treated with hot 

 NaOH as in sample B. The NH 4 OH treatment caused a rearrangement 

 of the molecule or stabilization, for on acid hydrolysis glucose number 

 was 10.25 as compared to zero with sample B. Optical activity was re- 

 duced but not entirely lost. Immunological tests, although decreased, 

 were not as low as in sample B, with the exception of the combining 

 equivalent and that was the lowest of all samples, 0.9 unit. The acid 

 from vacuum distillation was higher than in sample D, indicating a 

 splitting off of the terminal carboxyl radicals. 



At this stage of our investigation certain inferences may be made: 

 (1) An antigen has been prepared from a Type I polysaccharide which 

 in our opinion is non-protein in nature for the following reasons: (a) 

 treatment with hot NaOH (N/lO) and then NH 4 OH results in a prod- 

 uct more highly antigenic than the original preparation, and (b) all 

 well-recognized protein tests are negative including a test for sulfur 

 with a large sample of material. (2) This antigen may be considered 



by dilution with 2 volumes of 1 : 1 alcohol-ether, washed repeatedly with alcohol, 

 alcohol-ether and dried. 



Sample D was made from the original material (A) by treatment with 

 NH.OH as in C. 



Sample E was prepared from D by the same procedure as B, in other words 

 heating at 100°C. in the Arnold sterilizer in N/10 NaOH for 30 minutes. 



