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Abstract
Enzymic casein hydrolysate, which is a suitable nitrogenous basis of the medium for alph-toxin production by Clostridium welchii (perfringens), was fractionated by column chromatography on Sephadex G25 gel, using ammoniacal buffer. Combined peptidic fractions were able to support microbial growth, though toxin production was minimal. Toxinogenic substances were retained on the gel and could be eluted with weak acetic acid. This active substance represented about 0·5% of the original hydrolysate and consisted of a mixture of low molecular weight peptides with free amino acids and a mineral portion (calcium, magnesium and iron). When column chromatography of enzymic casein hydrolysate was done with Sephadex G25 and 0·05 m-ammonium hydrogen carbonate buffer, toxinogenic substances were not retained on the gel. The procedure resulted in a group separation of peptides according to their molecular weight and simultaneously an adsorption effect was observed of aromatic amino acids on the gel. Four fractions were obtained exhibiting different effects on growth and toxinogenesis by C. welchii.
The fraction containing the largest molecules and the major part of total nitrogen (90% approx.) proved to stimulate microbial growth, though only minimal α-toxin production. The other three low molecular fractions exhibit a stimulating effect on α-toxin production; their amino acid compositions were different.
The most active of the low molecular weight fractions contained about 1·8% of the initial nitrogen and could be fractionated with DEAE-Sephadex A25 into a further four groups of which the most remarkable was peptide-bound tyrosine.
Gel chromatography on Sephadex columns resulted in an unequal distribution of Fe ions among the fractions; this was allowed for in performing comparative microbiological tests.
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