Abstract
In an earlier paper, 1 there was described the separation, from meningococcus autolysates, of fractions exhibiting marked type-specificity toward precipitin-containing sera. It is now possible to report the preparation of the Type I specific substance (SSS) in a much higher state of purification.
Twenty liter lots of 3 week cultures of recently isolated strains of Type I meningococcus in hormone broth are acidified with acetic acid to pH 4.0. The SSS has a marked affinity for the acid-precipitable protein, the so-called “P” fraction, and about one-half of the amount present is carried down with the latter under the above conditions. If the broth is diluted 10-fold with tap water before acidification, almost all the SSS is adsorbed, but there is a loss in selectivity. This point is undergoing further investigation.
The “P” fraction is washed with distilled water, suspended in 1 liter of the same solvent and dissolved at pH 7.0 with the aid of NaOH. The bulk of the protein is then precipitated with acetic acid at pH 4.8 and centrifuged off. The precipitate is washed with distilled water and again submitted to the process of solution at pH 7.0 and precipitation at pH 4.8. The combined supernatant fluids, which contain almost all of the SSS, are concentrated in vacuo to the smallest practicable volume and precipitated with 5 volumes of alcohol. After standing overnight, the precipitate is centrifuged off and dried in vacuo with concentrated H2SO4. It is then dissolved in 50 to 100 cc. of distilled water with the aid of NaOH, and freed of the remainder of the protein by 0.7 saturation with (NH4)2SO4. The latter is removed by dialysis, 10% of sodium acetate is added and the SSS is precipitated as a yellowish gum by 4 volumes of ethyl alcohol. Further purification is achieved by repeated precipitation by alcohol in the presence of sodium acetate and through precipitation with basic lead acetate or uranyl nitrate. Finally, the SSS is precipitated as a white powder by 10 volumes of absolute alcohol, from aqueous solution containing a small amount of sodium acetate. In favorable cases the yield from 20 liters of broth is 350 mg.
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