Purification of two Clostridium bacteriocins by procedures appropriate to hydrophobic proteins.
Two clostridocins distinguishable by their different modes of action on Clostridium pasteurianum have been isolated, namely, butyricin 7423 found in cultures of Clostridium butyricum NCIB 7423 and perfringocin 11105 produced by Clostridium perfringens type A, NCIB 11105. Both were trypsin-susceptible proteins which were soluble in concentrated aqueous ethanol and were able to bind large amounts of the nonionic detergent Triton X-100. In the presence of Triton X-100, butyricin 7423 behaved as a hydrophobic protein in being concentrated in the polyethylene glycol layer of a three-phase partition system of dextran-Ficoll-polyethylene glycol. Their capacity to bind Triton X-100 was exploited in a purification procedure applicable to both bacteriocins. After aqueous ethanol extraction of an ammonium sulfate-precipitated fraction (and, in the case of the perfringocin, a heat-treatment step), a bacteriocin-Triton X-100 adduct was purified by gel filtration through Sepharose 6B. The bacteriocin was then freed of Triton X-100 by chromatography on Sephadex LH-20. Samples of butyricin 7423 purified in this way from different sources contained variable amounts of carbohydrate. Yet sodium dodecyl sulfate-gel electrophoresis revealed the existence of a polypeptide component of 32,500 daltons (+/-10%), which displayed the biological activity of butyricin 7423 in the absence of any detectable associated carbohydrate (or lipid). Preparations of perfringocin 11105 contained no carbohydrate or lipid and migrated in sodium dodecyl sulfate-gel electrophoresis as a single protein component of 76,000 daltons (+/-10%). It was concluded that both bacteriocins behave as amphiphilic proteins, and some implications of this finding are considered.
D001430:Bacteriocins / Q000096:biosynthesis / Q000302:isolation & purification*; D003013:Clostridium / Q000378:metabolism; D008722:Methods; D011506:Proteins / Q000302:isolation & purification*
D001430:Bacteriocins; D011506:Proteins