SUMMARYFactor I of the anthrax toxin was isolated and showed one major component in the ultracentrifuge and on paper electrophoresis; it contained less than 5.5% of extraneous antigens detectable by serological precipitation in gels. The final preparation contained all the usual amino acids (N = 10*1%) and some carbohydrate (6 yo, calculated as glucose) and phosphorus (0.7 yo). The most striking aspects of its analysis were a high ash (10-13y0) and a light absorption a t 260 mp. The high ash was not due to one element but to a highly variable metal content (mainly Ca, Mg, Ni, Cu) indicating a powerful and indiscriminate chelating action of factor I. This chelating action might have been due to the chemical entity which absorbed light a t 260 mp and which was not RNA or DNA.The final preparation of factor I was not toxic when injected alone but when mixed with purified factor I1 it evoked oedema in the skin of a rabbit and killed mice. However, the concentration of this mixture which killed mice formed a much larger skin reaction in rabbits than a comparable dose (based on mouse LD50) of either crude toxin or a mixture of crude factors I and 11. An investigation of this fact led to the demonstration and partial purification of a third factor (111) of the anthrax toxin which: (1) was different serologically from factors I and 11; (2) was present in anthrax toxin produced in vivo; (3) was non-toxic when injected alone; (4) was lethal for mice when mixed with factor I1 but not with factor I ; ( 5 ) increased the lethality of mixtures of factors I and I1 for mice and decreased their capacity to produce oedema in the skin of rabbits. A mixture of factors I, I1 and I11 showed synergic action in toxicity tests in mice; the mixture killed guinea pigs which showed signs of oligaemic secondary shock (as did guinea pigs killed by anthrax infection).

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By growing Aspergillus parasztzcus (C.M.I. 15957) under controllcd conditions on sterilized peanuts an average of 265 nig. of aflatoxin per kilogram of peanuts has been produced and subsequently isolated. Other strains from the A . fluz~usoryzae group gave lower yields of aflatoxin and one such strain gave aflatoxin froni which certain normal coinponents were absent. The aflatoxin produced on sterilized peanuts by any particular strain of A . fivus-oryzue was shown by thin-layer chromatography to contain the same major components as were produced by that strain on unsterilized whole peanuts.

SUMMARY:Highly labile factors (I and 11) of anthrax toxin were purified from the toxic plasma of guinea-pigs just dead from anthrax by fractionation methods which involved the xninimuxn of inaetivation. The final preparations of factors I and 11, which still contained constituents of guinea-pig plasma, were toxic when injected together but innocuous when injected separately.The specific, lethal and oedema-producing toxin of Bm*Zlw ar&hraciS found in the plasma of guinea-pigs dying of anthrax (Smith, Keppie & Stanley, 1955) has two components which form a synergic mixture (Smith et al. Further purification of these factors was hampered by their extreme lability. The present paper describes fractionations whereby they were considerably purified without appreciable loss of toxicity. A brief report of some of this work has appeared elsewhere (Stanley & Smith, 1958; Sargeant & Smith, 1958). METHODSAnthrax toxin produced in infected guinea-pigs was obtained as described by Smith et aZ. (1055). It was imperative that the plasma of guinea-pigs should be collected within a few minutes of death from anthrax. When delay occurred here, or in the subsequent removal of bacteria, the tendency for factor I to lose activity and to aggregate and become insoluble during subsequent fractionation was more pronounced.This was described by Peterson & Sober (1956). It was made in the laboratory from I.C.I. solka floc S.W. 40.B or bought from Eastman Kodak Co.Assay of factors I and I I of arzthrax toxin. Serial descending dilutions (0.1 ml.) of the test sample (factor I or factor 11) were injected intradermally into the shaven side of a rabbit, immediately after mixing each dilution with a fixed amount (0.1 ml.) of the complementary factor (either crude factor I1 or crude factor I; for preparation see below) a t a concentration which just failed to produce a skin lesion when 0.2 ml. was injected alone. The oedematous lesions formed by these mixtures of factors I and I1 were observed 16 hr. after injection and the null point, i.e. the first dilution of the test sample not giving a positive reaction, was noted as a measure of factor I or factor 11. The assay would detect twofold differences in activity.