Tuberculinic acid

The chemistry of tuberculinic acid (the nucleic acid of the tubercle bacillus) was investigated by Brown and Johnson. The acid was purified by conversion to the copper salt. Distillation with hydrochloric acid yielded small amounts of furfural, indicating the presence of only a trace of pentose in the residue. Levulinic acid was identified, and it was thought on this evidence that the sugar associated with the acid was a hexose. Tuberculinic acid is unique in that it does not contain uracil, has a low pentose content and contains an unusual pyrimidine derivative. The tuberculinic acid was considered to be more nearly related to deoxyribonucleic acid than to ribonucleic acid. ... 

Methylcytosine was first found in tuberculinic acid obtained from tubercule bacilli. Later it was isolated from thymus DNA and found to be a minor pyrimidine constituent of mammalian DNA and a major pyrimidine compound in wheat germ DNA. However, contrary to the original reports, 5-methylcytosine could not be found in bacterial DNA, including tubercule bacilli. 

The presence of small amounts of 5-methyl cytosine in desoxyiibo-nucleic acid has been noted already. It has been isolated both as the nucleotide and the nucleoside. This pyrimidine was discovered some years ago in acid hydrolysates of tuberculinic acid. ... 

The most common carrier proteins in use today are keyhole limpet hemocyanin (KLH MW 4.5 X 105 to 1.3 X 107), BSA (MW 67,000), aminoethylated (or cationized) BSA (cBSA), thyroglobulin (MW 660,000), ovalbumin (OVA MW 43,000), and various toxoid proteins, including tetanus toxoid and diphtheria toxoid. Other proteins occasionally used include myoglobin, rabbit serum albumin, immunoglobulin molecules (particularly IgG) from bovine or mouse sera, tuberculin purified protein derivative, and synthetic polypeptides such as poly-L-lysine and poly-L-glutamic acid. 

Figure 6.4 Experimental curves for equilibrium gel filtration, (a) The 1-mL tuberculin syringe was incubated in 109-pAf[14C]valine (O) or 109-/zM[14C]valine and 4-mM ATP ( ). Then 100 fjL of a solution of 26-fiM valyl-tRNA synthetase was added to the same solution. Stoichiometries of 0.8 and 1.1, respectively, were found for the binding of the amino acid. Note the return to baseline between the peak and the trough— the mark of a good equilibrium gel filtration experiment, (b) An artifact-induced double peak obtained from the binding of [y-32P]ATP and valine to the enzyme. Some of the labeled ATP hydrolyzed to [32P]orthophosphate, which traveled down the column faster than the fy-32P]ATP did.

Comprehensive reviews of the earlier investigations have been published by Cornet and by Wells, Dewitt and Long. A recent review of the composition of tuberculin and the proteins of acid-fast bacilli has been published by Seibert. Andersonhas summarized the chemical knowledge of the lipids of M. tuberculosis. 

These results were confirmed in the main by Dorset and Henley. A polysaccharide was obtained from Long s synthetic medium after growth of M. tuberculosis, human strain. This polysaccharide showed a positive biuret test for protein, and underwent hydrolysis only with difficulty. D-Arabinose and D-mannose were identified in the hydrolysate. No glycuronic acid could be detected. On hydrolysis, the D-arabi-nose seemed to be liberated before the other constituents. It was concluded from the biological tests that the active principle of tuberculin concerned in eliciting the skin reaction was a protein. This work was repeated with polysaccharides derived from the culture medium of the bovine and avian strains of organisms, and very similar results were obtained. D-Arabinose and D-mannose were also identified in those polysaccharides. 

Dienes and Schonheit showed that the filtrates of cultures grown on Long s synthetic medium contained two distinct antigenic fractions, separable by acid precipitation, together with carbohydrate precipitable material. Seibert and Munday confirmed the fact that the tuberculin skin reaction was due to proteins present in tuberculin, since fractional hydrolysis of the crude tuberculin precipitate afforded a very... 

Nucleic acid also was found in association with the tuberculin protein. Spiegel-Adolf and Seibert investigated the problem spectrographically, and found that the tuberculin protein ( P.P.D. ) precipitated by trichloroacetic acid exhibited an absorption band at 2650-2670 A. This band was identical with that displayed by deoxyribonucleic acid. Precipitation of the tuberculin protein by ammonium sulfate afforded a product which did not display an absorption band in the ultraviolet region of the spectrum. The substance had a lower phosphorus content than that of products obtained by trichloroacetic acid precipitation. The biological activity was not impaired in any way. 

A separation of the nucleic acid, polysaccharide and protein fractions of tuberculin was effected by the Tiselius electrophoretic technique, which was developed for large scale work in this field by Seibert and Watson. The polysaccharide was relatively immobile and thus was easily removed from the protein and nucleic acid. At pH 5.0 (or less), the nucleic acid and protein traveled in the electrophoretic tube as a single component. At higher pH values, the two tended to move independently. It was probable that some of the protein and nucleic acid was present as nucleoprotein. 

Ascorbic Acid and Tuberculin-Sensitivity in Guinea Pigs. 72... 

---