Molecular Weight, Electrochemical and Biological Properties of Tuberculin Protein and Polysaccharide Molecules

Studies have been made by means of sedimentation in the ultracentrifuge, and by diffusion and electrophoresis, to determine the molecular weights and homogeneity of the tuberculin protein and polysaccharide molecules as found in their natural state in the unchanged filtrates from culture media after growth of tubercle bacilli. These results have been compared with data obtained on fractions isolated by chemical procedures from them or from old tuberculin. By means of electrophoresis in the Tiselius apparatus it was possible to separate the protein from the polysaccharide, as these two fractions occur naturally in the original culture medium filtrates of acid-fast bacilli. The protein from the bovine strain of bacillus proved to be homogeneous in sedimentation (S(20) = 1.6), diffusion (D(20) = 12.0) and electrophoresis, with a molecular weight of about 10,000. The tuberculin polysaccharide isolated in electrophoresis appeared to be practically the same in sedimentation and in precipitin reaction as the polysaccharide isolated by chemical procedure. The latter proved to be homogeneous in sedimentation (S(20) = 1.6) and diffusion (D(20) = 11.0) with a molecular weight of about 9000. A practically homogeneous protein was isolated from the culture filtrate of the human tubercle bacillus H 37 by fractional ammonium sulfate precipitation, with a molecular weight of 32,000 (S(20) = 3.3; D(20) = 8.2). It was electrochemically homogeneous, with an isoelectric point at pH 4.3 and an isoionic point at pH 4.7. It could be dried or frozen with no loss in homogeneity. It was highly antigenic in the precipitin reaction and produced the anaphylactic type of local skin reaction in tuberculous guinea pigs, in contrast to the true tuberculin type of reaction caused by a purified PPD fraction. Furthermore death resulted in tuberculous guinea pigs from intracutaneous injection of exceptionally small amounts. A protein with molecular weight of about 17,000 was isolated from the filtrate from cultures of the timothy bacillus. The nucleic acid originally occurring in old tuberculin (OT) seems to be responsible for the high electrochemical mobility observed. From OT and the PPD made from it, potent but non-antigenic molecules of 16,000 and 9000 weight and with a low content of nucleic acid were isolated. With increase in size these deviated much from the normal compact spherical shape, and aggregation was evident from the tendency toward gel formation. The smallest molecule (9000) was homogeneous (S(20) = 1.0; D(20) = 10.0) and had lost some tuberculin potency.