Abstract
In an effort to produce a potent tuberculin in a concentrated form and with the least possible chemical manipulation, Seibert 1 resorted to ultrafiltration, a method based on the conclusion from previous experiments that the active principle in tuberculin which is responsible for eliciting skin reactions in tuberculous guinea pigs is a water-soluble protein of the nature of an albumin. While this appears to be a promising method for concentrating the protein without incurring the possibility of denaturation, the end product is still complex since it retains all of the non-dialyzable constituent of the original filtrate among which are metaproteins, proteoses, and a large proportion of carbohydrate.
The aim of this investigation was to determine (1) whether the carbohydrate is linked up with the protein molecule in the form of a glucoprotein, and (2) if not, to prepare a protein fraction free from carbohydrate in order that its biological activity would not be masked by its presence. A method is outlined below for preparing such a fraction, to which we have given the designation of Human Tubercle Bacillus Protein MA-100 to conform with Johnson's scheme of nomenclature.
A culture of Tubercle bacilli (H-37) was grown on Long's synthetic medium and then filtered through a filter candle. To 11,000 cc. of this filtrate was added 7150 gm. (NH4)2SO4. The precipitated protein soon rose to the top of the vessel and after standing several hours it was separated from the liquid by filtering through a Büchner funnel. The precipitate was purified by 8 successive reprecipitations. The precipitate from the last operation was dissolved in 1000 cc. distilled water, filtered through paper, and to the clear solution was added 30 cc. of Rimington's 2 salt buffer mixture (pH 4.7).
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