Biochemical Studies of Bacterial Derivatives. X. Preparation of Human Tubercle Bacillus Protein MA-100. ∗

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).