Soluble Specific Substance of Pneumococcus Type III Possessing Properties Distinct from SSS III

Abstract

It has been recently demonstrated that the soluble specific substance of Pneumococcus type I, as originally prepared by Heidelberger, Goebel and Avery, represented a form of the polysaccharide lacking the acetyl group which, when present, conferred upon the substance distinct physico-chemical and immunological properties. 1 , 2 In the case of the type specific polysaccharide from Pneumococcus type III, purified according to the procedure described by the same authors, certain observations such as those of Ward 3 have led us to believe that an analogous alteration in chemical structure may have occurred during the chemical manipulations required for its purification. We have, therefore, attempted to prepare this material by a method which avoids insofar as possible the use of strong acids.

Six- to 8-day cultures of Pneumococcus type III in dextrose phosphate broth were concentrated over a boiling water bath to one-tenth of the original volume. The concentrate was precipitated several times with about 1.2 volumes of alcohol. Proteins were precipitated by careful addition of 1N acetic acid, until maximal precipitation was attained. Neutralization and reprecipitation with the acid was twice repeated.

After the last acid precipitation, the supernatant was made alkaline and precipitated with alcohol. The precipitate was dissolved in H₂O and 2 volumes of saturated ammonium sulfate added, the scanty precipitate discarded and the sulfate removed by dialysis against distilled water. The solution of polysaccharide in the dialyzing sack was precipitated with 1.2 volumes of alcohol. After resolution in H₂O and precipitation with acetone, it was washed with alcohol and ether, and dried in vacuo.