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. 1974 Sep;28(3):374–382. doi: 10.1128/am.28.3.374-382.1974

Clostridium botulinum Type E Toxin: Effect of pH and Method of Purification on Molecular Weight

Henry S Sacks 1, Scott V Covert 1
PMCID: PMC186729  PMID: 4609265

Abstract

The toxin of Clostridium botulinum type E was isolated from intact cells and from toxic culture filtrates by column chromatography at three pH values, 4.5, 5.3, and 6.0. At pH 6.0 and 5.3, the isolated toxin was in a form with a molecular weight (MW) of 86,000. This toxin was homogeneous on polyacrylamide gel electrophoresis and gel filtration and had an optical density ratio, 280 nm/260 nm, greater than 2.0. It did not dissociate at higher pH levels, but was dissociated into nontoxic components of approximately 12,000 MW when reduced and alkylated in the presence of 6 M guanidine hydrochloride. At pH 4.5, smaller amounts of an impure toxic moiety with a MW of 12,000 were found. After storage for 6 months, the 86,000-MW moiety had lost 60% of its lethality. Gel filtration revealed that the bulk of the toxicity was associated with a component having a MW of 150,000. Toxic components with MW of 12,000 and over 200,000 were also found. The toxin appears to polymerize or aggregate when in a pure form, so that most, if not all, of the MW previously reported for the toxin may belong to different polymers of a monomer with a MW of 12,000 or less. Treatment of the 86,000-MW toxin with trypsin resulted in an 18- to 128-fold increase in lethality, but no detectable change in MW.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Andrews P. The gel-filtration behaviour of proteins related to their molecular weights over a wide range. Biochem J. 1965 Sep;96(3):595–606. doi: 10.1042/bj0960595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. DasGupta B. R., Sugiyama H. A common subunit structure in Clostridium botulinum type A, B and E toxins. Biochem Biophys Res Commun. 1972 Jul 11;48(1):108–112. doi: 10.1016/0006-291x(72)90350-6. [DOI] [PubMed] [Google Scholar]
  3. Emodi A. S., Lechowich R. V. Chromatographic purification of the toxin of type E Clostridium botulinum. Biochem Biophys Res Commun. 1969 Jun 27;35(6):788–795. doi: 10.1016/0006-291x(69)90692-5. [DOI] [PubMed] [Google Scholar]
  4. Fish W. W., Mann K. G., Tanford C. The estimation of polypeptide chain molecular weights by gel filtration in 6 M guanidine hydrochloride. J Biol Chem. 1969 Sep 25;244(18):4989–4994. [PubMed] [Google Scholar]
  5. GERWING J., DOLMAN C. E., BAINS H. S. ISOLATION AND CHARACTERIZATION OF A TOXIC MOIETY OF LOW MOLECULAR WEIGHT FROM CLOSTRIDIUM BOTULINUM TYPE A. J Bacteriol. 1965 May;89:1383–1386. doi: 10.1128/jb.89.5.1383-1386.1965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. GERWING J., DOLMAN C. E., KO A. MECHANISM OF TRYPTIC ACTIVATION OF CLOSTRIDIUM BOTULINUM TYPE E TOXIN. J Bacteriol. 1965 May;89:1176–1179. doi: 10.1128/jb.89.5.1176-1179.1965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. GERWING J., DOLMAN C. E., REICHMANN M. E., BAINS H. S. PURIFICATION AND MOLECULAR WEIGHT DETERMINATION OF CLOSTRIDIUM BOTULINUM TYPE E TOXIN. J Bacteriol. 1964 Jul;88:216–219. doi: 10.1128/jb.88.1.216-219.1964. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gerwing J., Dolman C. E., Kason D. V., Tremaine J. H. Purification and characterization of Clostridium botulinum type B oxin. J Bacteriol. 1966 Feb;91(2):484–487. doi: 10.1128/jb.91.2.484-487.1966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hjertén S., Jerstedt S., Tiselius A. Some aspects of the use of "continuous" and "discontinuous" buffer systems in polyacrylamide gel electrophoresis. Anal Biochem. 1965 May;11(2):219–223. doi: 10.1016/0003-2697(65)90008-4. [DOI] [PubMed] [Google Scholar]
  10. Kitamura M. Behaviour of toxin released from the cells of Clostridium botulinum type E. Biochem Biophys Res Commun. 1970 Aug 24;40(4):925–931. doi: 10.1016/0006-291x(70)90992-7. [DOI] [PubMed] [Google Scholar]
  11. Kitamura M., Sakaguchi S., Sakaguchi G. Dissociation of Clostridium botulinum type-E-toxin. Biochem Biophys Res Commun. 1967 Dec 29;29(6):892–897. doi: 10.1016/0006-291x(67)90305-1. [DOI] [PubMed] [Google Scholar]
  12. Kitamura M., Sakaguchi S., Sakaguchi G. Purification and some properties of Clostridium botulinum type-E toxin. Biochim Biophys Acta. 1968 Oct 21;168(2):207–217. doi: 10.1016/0005-2795(68)90144-x. [DOI] [PubMed] [Google Scholar]
  13. Kitamura M., Sakaguchi S., Sakaguchi G. Significance of 12S toxin of Clostridium botulinum type E. J Bacteriol. 1969 Jun;98(3):1173–1178. doi: 10.1128/jb.98.3.1173-1178.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. SAKAGUCHI G., SAKAGUCHI S., IMAI N. COMPARATIVE GEL FILTRATION OF TOXIN PRECURSOR AND TRYPSIN-ACTIVATED TOXIN OF CLOSTRIDIUM BOTULINUM TYPE E. J Bacteriol. 1964 Feb;87:401–407. doi: 10.1128/jb.87.2.401-407.1964. [DOI] [PMC free article] [PubMed] [Google Scholar]

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