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An immunogenic and skin-reactive protein called P64 was purified from Sauton zinc-deficient culture filtrate of Mycobacterium bovis BCG by using successively hydrophobic chromatography on phenyl-Sepharose, ion exchange on DEAE-Sephacel, and molecular sieving on Sephadex G-200. The final P64 preparation was found to be homogeneous based on several analyses. Protein P64 was a constituent of BCG cells since it was present in soluble cellular extract from normally grown BCG cells. It represented 8 to 9% of the soluble proteins of the extract and appeared as the major soluble protein antigen of BCG. This protein was found to have a molecular weight of 64,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but in molecular sieving it eluted at a volume corresponding to a molecular weight of 246,000. An abnormal UV spectrum was observed for this protein. Its amino acid composition showed an abundance of acidic amino acids (or their amides). Aromatic amino acids represented only 3% of the total amino acid residues. The NH2-terminal amino acid sequence of this protein (10 amino acids) was determined. Its sugar content measured with the phenol-sulfuric acid test was lower than 0.3% (wt/wt). Isolated P64 was tested by various crossed-immunoelectrophoresis techniques and was shown to correspond to antigen 82 in the reference system for BCG antigens. The protein antigen P64 elicited a delayed cutaneous reaction in guinea pigs sensitized with either living or heat-killed BCG. Its potency in skin reaction was, respectively, two-and threefold that of the BCG purified protein derivative. The two types of sensitization used for skin test reactions promoted significant immunoglobulin G antibody production against the protein antigen P64 in guinea pigs 7 weeks after sensitization.

An alcohol dehydrogenase of broad specificity was purified 43-fold from extracts of Mycobacterium tuberculosis var. bovis (BCG) grown on Sauton medium. It was also present in cells grown in Dubos medium and Tween 80 and bovine serum albumin. The enzyme, which appeared to be soluble, acted as an oxidoreductase in the system butan-1-ol-NADP. It was eluted from Sephadex G-200, hydroxylapatite and DEAE-cellulose in a single peak. The molecular weight, as determined by gel filtration on Sephadex G-200, was about 75,000. Results of electrophoresis in sodium dodecyl sulphate-polyacrylamide gels were compatible with the existence of two subunits each of molecular weight 37,500. The optimum pH was about 8.5 when the enzyme catalysed the oxidation of butan-1-ol, and about 8.2 for the reverse reaction. The apparent Km was 0.125 mM for butyraldehyde and 0.22 M for butan-1-ol. The dehydrogenase activity was maintained after heat treatment (40 min at 55 degrees C) in the presence of 30% (W/V) glycerol, but was abolished by heating (40 min at 55 degrees C) in the presence of 0.1 M-EDTA. The activity of enzyme inactivated by heat and EDTA could be fully restored at room temperature in the presence of 2 mM-Zn2+.

Mycobacterium tuberculosis var. bovis (BCG) grown on Sauton medium normally forms a pellicle; in the absence of added Zn2+, however, the pellicle sank during incubation and the yield was only about 20% of normal. The Zn2+-starved bacteria were morphologically similar to normal bacteria and were still acid-fast at 7 d as well as 14 d. The Zn2+-starved bacteria had slightly higher free lipid and phospholipid contents than normal; the content of hexoses was lower and proteins slightly lower. The deficient culture medium became opalescent and alkaline. Aspartate and ammonium ions accumulated. There was twice as much protein in deficient as in normal medium; moreover, a class of proteins precipitable at pH 4.5, which was hardly detectable in normal medium, was present in appreciable amounts of deficient medium. The content of aldehydes, measured with yeast alcohol dehydrogenase, was also doubled in deficient medium. Fractionation of acid-soluble aldehydes obtained from deficient medium after acid treatment of a bisulphite precipitate suggested the presence of several complex molecules bearing aldehyde groups. The need for Zn2+ in the medium may be explained by the presence in normal BCG of a Zn2+-requiring NADP-dependent alcohol dehydrogenase activity whose affinity for aldehydes is especially high.

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