Physical-chemical properties of precipitated and fractionated allergens from Mycobacterium bovis.

Seven allergens were prepared from cultures of Mycobacterium bovis. The culture filtrates were divided in half and each aliquot precipitated with either ammonium sulfate or trichloroacetic acid to prepare purified protein derivatives (PPD). These samples of PPD-bovis were analyzed for nitrogen, carbohydrate, RNA and DNA. Physical tests, such as: cellulose acetate electrophoresis, disc gel electrophoresis, ultracentrifugation, isoelectricfocusing and gel filtration were also performed. Precipitation with ammonium sulfate provided PPD's with less impurities than those precipitated with trichloroacetic acid although there was little difference in the allergenic activity of the products precipitated by either method. Several lots of PPD-bovis were fractionated by means of ultrafiltration and the fractions analyzed by many of the above chemical and physical tests as well as tests for amino acid composition and allergenic activity. The greatest amount of protein was found in the fractions with an apparent molecular weight in excess of 300,000. The carbohydrate was usually distributed in proportion to the amount of protein in each fraction while the nucleic acids were predominately found in the higher molecular weight fractions. The amino acid analysis and allergenic activity of the various molecular weight fractions provided data that would support the theory that the various fractions are polymers of a small molecular weight sub-unit. The number of amino acid residues were calculated and the minimum molecular weight of the tubercular protein estimated to be 8700. The estimated molecular weights of the fractions and the amino acid data concur with those previously reported for proteins from other mycobacteria.