Abstract
When a Type I pneumococcus antiserum is absorbed with the homologous soluble specific carbohydrate until no further precipitin reaction occurs with the latter, the absorbed serum still reacts type-specifically with a substance in pneumococcus autolysates in high dilution, as shown by Enders 1 and confirmed by Wadsworth and Brown 2 , 3 and by Sabin. 4 We have now succeeded in isolating this substance, called the “A substance” by Enders, from Type I pneumococcus broth cultures.
The initial steps in the preparation of the A substance are essentially those given by Heidelberger and Kendall 5 in their preparation of the soluble specific substance, SSS1, except that care is taken to maintain an acid reaction throughout. When, as in their procedure, the specific carbohydrate is precipitated with acetic acid at pH 3.4, the A substance remains in solution. We have found, contrary to Heidelberger and Kendall, that it is frequently not possible to obtain an appreciable precipitation of SSS1 at this point. This matter will be discussed below. At any rate, whether or not a precipitate is obtained, the A substance is present in the acid supernatant from which it is now precipitated by alcohol and purified by precipitation with copper chloride at pH 3.4 as noted. The excess copper is removed by repeated solution in 10% acetic acid and precipitation with alcohol. The yield varies from about 100 mg. to 600 mg, from 20 L of broth culture, depending on how much SSS1 is removed by precipitation with acetic acid.
The A substance prepared as outlined above is an extremely hygroscopic, white amorphous powder, soluble in the range pH 1-9. It contains no sulfur or phosphorus. As recorded in Table I, its elementary analysis, amino-nitrogen content (van Slyke) and specific rotation are practically identical with the SSS1 as prepared by Heidelberger, Goebel and Avery. 6
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