Abstract
The three components of the toxin of Bacillus anthracis, edema factor (EF), protective antigen (PA), and lethal factor (LF), were purified 197-, 156-, and 1,025- fold, with 38, 78, and 11% recovery, respectively. Each purified component was serologically active, distinct, and free from the other components. The purified EF produced edema when mixed with PA, and the purified PA was an active immunogen. The components did not appear to be simple proteins by spectrophotometric analysis. As they were purified, the pH range in which they were most stable narrowed, centering between pH 7.4 and 7.8. Heat readily destroyed the biological activity of the components but not their serological activity. The rat lethality test showed that, with a constant amount of LF and an increasing amount of PA, the time to death reached a minimum and then was extended. When an increasing amount of LF was added to a constant amount of PA, the time to death became shorter as more LF was added. The biological, immunological, and serological properties of the components were shown to vary independently with storage and extent of purification so that serological activity was not always directly correlated with biological activity. Evidence is presented that the components can exist in different molecular configurations or as aggregates, and that this property is influenced by the state of component purity and by the environment.
Full text
PDF![907](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/480a/252109/724a0e53ba7b/jbacter00401-0205.png)
![908](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/480a/252109/33ab36d94911/jbacter00401-0206.png)
![909](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/480a/252109/eba3557fc4ee/jbacter00401-0207.png)
![910](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/480a/252109/ee13f705d5f9/jbacter00401-0208.png)
![911](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/480a/252109/1d59752deb1c/jbacter00401-0209.png)
![912](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/480a/252109/9dd4ccbefdb0/jbacter00401-0210.png)
![913](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/480a/252109/4be11cc92955/jbacter00401-0211.png)
![914](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/480a/252109/3080c14c0e59/jbacter00401-0212.png)
![915](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/480a/252109/8118c35ead11/jbacter00401-0213.png)
![916](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/480a/252109/d026501fd1bf/jbacter00401-0214.png)
![917](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/480a/252109/af4efa3308a0/jbacter00401-0215.png)
![918](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/480a/252109/a47be5aa30a9/jbacter00401-0216.png)
Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- ALLISON A. C., HUMPHREY J. H. Estimation of the size of antigens by gel diffusion methods. Nature. 1959 Jun 6;183(4675):1590–1592. doi: 10.1038/1831590a0. [DOI] [PubMed] [Google Scholar]
- ANDREWS P. Estimation of molecular weights of proteins by gel filtration. Nature. 1962 Oct 6;196:36–39. doi: 10.1038/196036a0. [DOI] [PubMed] [Google Scholar]
- BEALL F. A., TAYLOR M. J., THORNE C. B. Rapid lethal effect in rats of a third component found upon fractionating the toxin of Bacillus anthracis. J Bacteriol. 1962 Jun;83:1274–1280. doi: 10.1128/jb.83.6.1274-1280.1962. [DOI] [PMC free article] [PubMed] [Google Scholar]
- BOOR A. K., TRESSELT H. B. Protection of sheep against anthrax by immunization with an antigen prepared in vitro. Am J Vet Res. 1955 Jul;16(60):425–428. [PubMed] [Google Scholar]
- DEARMON I. A., Jr, KLEIN F., LINCOLN R. E., MAHLANDT B. G., FERNELIUS A. L. Immunological studies of anthrax. I. An index to determine quantitative immunization. J Immunol. 1961 Sep;87:233–239. [PubMed] [Google Scholar]
- Fish D. C., Lincoln R. E. Biochemical and biophysical characterization of anthrax toxin. Fed Proc. 1967 Sep;26(5):1534–1538. [PubMed] [Google Scholar]
- Fish D. C., Lincoln R. E. In vivo-produced anthrax toxin. J Bacteriol. 1968 Mar;95(3):919–924. doi: 10.1128/jb.95.3.919-924.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
- HAINES B. W., KLEIN F., LINCOLN R. E. QUANTITATIVE ASSAY FOR CRUDE ANTHRAX TOXINS. J Bacteriol. 1965 Jan;89:74–83. doi: 10.1128/jb.89.1.74-83.1965. [DOI] [PMC free article] [PubMed] [Google Scholar]
- HARRIS-SMITH P. W., SMITH H., KEPPIE J. Production in vitro of the toxin of Bacillus anthracis previously recognized in vivo. J Gen Microbiol. 1958 Aug;19(1):91–103. doi: 10.1099/00221287-19-1-91. [DOI] [PubMed] [Google Scholar]
- MOLNAR D. M., ALTENBERN R. A. ALTERATIONS IN THE BIOLOGICAL ACTIVITY OF PROTECTIVE ANTIGEN OF BACILLUS ANTHRACIS TOXIN. Proc Soc Exp Biol Med. 1963 Nov;114:294–297. [PubMed] [Google Scholar]
- Mahlandt B. G., Klein F., Lincoln R. E., Haines B. W., Jones W. I., Jr, Friedman R. H. Immunologic studies of anthrax. IV. Evaluation of the immunogenicity of three components of anthrax toxin. J Immunol. 1966 Apr;96(4):727–733. doi: 10.21236/ad0640357. [DOI] [PubMed] [Google Scholar]
- RUSSELL W. R. The physiology of memory. Proc R Soc Med. 1958 Jan;51(1):9–15. [PMC free article] [PubMed] [Google Scholar]
- SARGEANT K., STANLEY J. L., SMITH H. The serological relationship between purified preparations of factors I and II of the anthrax toxin produced in vivo and in vitro. J Gen Microbiol. 1960 Feb;22:219–228. doi: 10.1099/00221287-22-1-219. [DOI] [PubMed] [Google Scholar]
- SMITH H., GALLOP R. C. The chemical basis of the virulence of Bacillus anthracis. VI. An extracellular immunising aggressin isolated from exudates of infected guinea-pigs. Br J Exp Pathol. 1956 Apr;37(2):144–155. [PMC free article] [PubMed] [Google Scholar]
- SMITH H., KEPPIE J. Observations on experimental anthrax; demonstration of a specific lethal factor produced in vivo by Bacillus anthracis. Nature. 1954 May 8;173(4410):869–870. doi: 10.1038/173869a0. [DOI] [PubMed] [Google Scholar]
- SMITH H., KEPPIE J., STANLEY J. L. The chemical basis of the virulence of Bacillus anthracis. V. The specific toxin produced by B. Anthracis in vivo. Br J Exp Pathol. 1955 Oct;36(5):460–472. [PMC free article] [PubMed] [Google Scholar]
- SMITH H., STANLEY J. L. Purification of the third factor of anthrax toxin. J Gen Microbiol. 1962 Nov;29:517–521. doi: 10.1099/00221287-29-3-517. [DOI] [PubMed] [Google Scholar]
- SMITH H., TEMPEST D. W., STANLEY J. L., HARRIS-SMITH P. W., GALLOP R. C. The chemical basis of the virulence of Bacillus anthracis. VII. Two components of the anthrax toxin: their relationship to known immunising aggressins. Br J Exp Pathol. 1956 Jun;37(3):263–271. [PMC free article] [PubMed] [Google Scholar]
- SMITH H. The use of bacteria grown in vivo for studies on the basis of their pathogenicity. Annu Rev Microbiol. 1958;12:77–102. doi: 10.1146/annurev.mi.12.100158.000453. [DOI] [PubMed] [Google Scholar]
- STANLEY J. L., SARGEANT K., SMITH H. Purification of factors I and II of the anthrax toxin produced in vivo. J Gen Microbiol. 1960 Feb;22:206–218. doi: 10.1099/00221287-22-1-206. [DOI] [PubMed] [Google Scholar]
- STANLEY J. L., SMITH H. Purification of factor I and recognition of a third factor of the anthrax toxin. J Gen Microbiol. 1961 Sep;26:49–63. doi: 10.1099/00221287-26-1-49. [DOI] [PubMed] [Google Scholar]
- STANLEY J. L., SMITH H. The three factors of anthrax toxin: their immunogenicity and lack of demonstrable enzymic activity. J Gen Microbiol. 1963 May;31:329–337. doi: 10.1099/00221287-31-2-329. [DOI] [PubMed] [Google Scholar]
- STRANGE R. E., BELTON F. C. Studies on a protective antigen produced in vitro from Bacillus anthracis: purification and chemistry of the antigen. Br J Exp Pathol. 1954 Apr;35(2):153–165. [PMC free article] [PubMed] [Google Scholar]
- STRANGE R. E., THORNE C. B. Further purification studies on the protective antigen of Bacillus anthracis produced in vitro. J Bacteriol. 1958 Aug;76(2):192–202. doi: 10.1128/jb.76.2.192-202.1958. [DOI] [PMC free article] [PubMed] [Google Scholar]
- TEMPEST D. W., SMITH H. The effect of metabolite analogues on growth of Bacillus anthracis in the guinea pig and on the formation of virulence-determining factors. J Gen Microbiol. 1957 Dec;17(3):739–749. doi: 10.1099/00221287-17-3-739. [DOI] [PubMed] [Google Scholar]
- THORNE C. B., BELTON F. C. An agar-diffusion method for titrating Bacillus anthracis immunizing antigen and its application to a study of antigen production. J Gen Microbiol. 1957 Oct;17(2):505–516. doi: 10.1099/00221287-17-2-505. [DOI] [PubMed] [Google Scholar]
- THORNE C. B., MOLNAR D. M., STRANGE R. E. Production of toxin in vitro by Bacillus anthracis and its spearation into two components. J Bacteriol. 1960 Mar;79:450–455. doi: 10.1128/jb.79.3.450-455.1960. [DOI] [PMC free article] [PubMed] [Google Scholar]
- WADDELL W. J. A simple ultraviolet spectrophotometric method for the determination of protein. J Lab Clin Med. 1956 Aug;48(2):311–314. [PubMed] [Google Scholar]
- WRIGHT G. G., HEDBERG M. A., SLEIN J. B. Studies on immunity in anthrax. III. Elaboration of protective antigen in a chemically defined, non-protein medium. J Immunol. 1954 Apr;72(4):263–269. [PubMed] [Google Scholar]