Isolation and purification of anaphylactically active polysaccharide from human tubercle bacilli.

In an earlier paper (Y. Yamamura et al., Am. Rev. Respirat. Diseases 91:839, 1965; I. Azuma et al., Am. Rev. Respirat. Diseases, in press), we reported the isolation and purification of a polysaccharide from a culture filtrate of human tubercle bacilli Aoyama B strain. The polysaccharide was shown to have anaphylactic activity in guinea pigs which were sensitized by heatkilled tubercle bacilli. This polysaccharide was composed of arabinose and a small amount of galactose. This note describes the extraction and purification of polysaccharide from defatted cells of human tubercle bacilli Aoyama B strain. The polysaccharide was shown to have anaphylactic activity in sensitized guinea pigs. Bacterial cells which were defatted by repeated extraction with ether-alcohol (1:1) and chloroform-methanol (1:1) were extracted with 1 N NaOH solution (100 g of cells per 2 liters of 1 N NaOH) at 70 C for 12 hr with stirring. The mixture was centrifuged at 10,000 rev/min for 60 min, and the supernatant fluid was neutralized with acetic acid. After centrifugation, the supernatant fluid was dialyzed against running water for 4 days and was concentrated to half volume. To the concentrate, 5 volumes of ethyl alcohol were added, and crude, precipitated polysaccharide was obtained by centrifugation and was designated "AB" fraction. The AB fraction was dissolved in a small amount of water and was separated into five fractions by fractional precipitation with ethyl alcohol. In the present experiments, two fractions, designated AB-66 and AB-80, were examined. AB-66 and AB-80 fractions were obtained by the addition of ethyl alcohol to a final concentration of 50 to 66% and 75 to 80%, respectively. Both AB-80 and AB-66 fractions contained a small amount of protein. The purification of AB-80 and AB-66 fractions was carried out by the following procedure. After the repeated fractional precipitation with ethyl alcohol and acetone, the fractions were chromatographed on a column of ion-exchange resin (Dowex 50, H+ form) and were eluted with water or 0.2 M Na2HPO4 solution. The water eluant, which was designated AB-80A fraction, was further chromatographed on a column of diethylaminoethyl (DEAE) cellulose and was eluted with water, 0.2 M NaH2PO4, or 0.1 N NaOH solution. The eluants were recovered by the addition of ethyl alcohol and were designated AB-8OAa, AB-8OAb, and AB-8OAc, respectively. The AB-8OAa fraction, which was eluted with water, was loaded on columns of Sephadex G-75 and G-200 and eluted with 0.5 M NaCi solution. AB-8OAa and AB-66Aa fractions purified in this way did not contain protein or nucleic acid. The intradermal injection of AB-66Aa and AB-8OAa fractions in 10-ug doses did not elicit a tuberculin reaction in a tuberculous patient. Precipitation tests showed that AB-66Aa and AB-8OAa fractions reacted with rabbit antitubercle sera in dilutions as high as 1:512,000. The test for analphylactic activity was examined in guinea pigs which were immunized with heat-killed tubercle bacilli in Freund adjuvant, by use of methods described previously (Y. Yamamura et al., Am. Rev. Respirat. Diseases 91:839, 1965). The strong anaphylactic activity in sensitized guinea pigs was found in AB-8OAa fraction. The sugar components of AB-66Aa and AB8OAa fractions were analyzed by gas-liquid chromatography. After methanolysis, trimethylsilyl derivatives of methyl glycosides were prepared by the methods of C. C. Sweeley et al. (J. Am. Chem. Soc. 85:2497, 1963) with some modifications. Gas-liquid chromatographic analysis of sugar derivatives was carried out by the methods of Sweeley et al. with the following as column packings: 5% of SE 52 on Shimalite W, 60 to 80 mesh, and 15% of polyethylene glycol succinate on Chromosorb W, 60 to 80 mesh. As shown in Table 1, AB-8OAa was composed of arabinose and mannose, whereas AB-66Aa consisted chiefly of mannose with a small amount of arabinose. Details of chemical structure of AB-66Aa and AB-8OAa fractions are being investigated in our