Abstract
In 1917 Dochez and Avery 1 showed that there was contained in filtrates from pneumococcus cultures and in the body fluids of experimentally infected animals and of patients suffering from pneumonia, a soluble substance which reacts specifically in anti-pneumococcus serum of the homologous type. This substance, which was found to be thermostable, precipitable by alcohol or acetone, non-dialyzable, and not digested by trypsin, is now being subjected to a more intensive chemical study.
Eight-day, autolyzed cultures of Type II Pneumococcus in phosphate broth were concentrated to 1/15 volume and precipitated with 1.2 volumes of alcohol. The precipitate, centrifuged at high speed, yields a compact middle layer containing the specific soluble substance. By repeated fractionation with alcohol or acetone, first in neutral, then in dilute acetic acid solution, followed by repeated fractional precipitation with ammonium sulfate and final dialysis, about 1 gm. of a highly purified preparation was obtained for each 75 liters of culture used.
In its present state of purity the specific soluble substance is amorphous and yields a viscous solution in water. A 1 per cent. solution gives no biuret test, yields no precipitate with phosphotungstic acid, mercuric chloride, or neutral lead acetate, gives a faint haze with tannic acid, and is precipitated by basic lead acetate. At a dilution of 1:1,500,000 it still gives the Molisch reaction and yields a precipitate with Type II immune serum. [a]D is + 58.7°; N, 1.2 per cent.; P, trace; S, none; C, 46.2 per cent.; H, 6.1 per cent. Hydrolysis yielded 79 per cent. of reducing sugars, of which glucose was identified by the melting point and optical rotation of its phenylosazone. Earlier preparations containing more nitrogen and yielding less reducing sugars on hydrolysis were not specific at as high dilutions.
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