Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1969 Jul;113(3):489–499. doi: 10.1042/bj1130489

Cleavage of bacterial flagellin with cyanogen bromide. Chemical and physical properties of the protein fragments

C R Parish 1,*, G L Ada 1,*
PMCID: PMC1184691  PMID: 5807209

Abstract

1. Flagellin, isolated from the flagella of Salmonella adelaide, was shown by various criteria to be a pure protein. It had a molecular weight of about 40000 and contained three methionine, six tyrosine, 11 arginine and 25 lysine residues/mol., of which 11 of the lysine residues were present as ∈-N-methyl-lysine. 2. After treatment of flagellin with cyanogen bromide in formic acid, four main fragments (A, B, C and D) were obtained, with as many as six minor components that represented partial degradation products. The major fragments were estimated by amino acid analysis to have molecular weights of about 18000 for fragment A, 12000 for fragment B, 5500 for fragment C and 4500 for fragment D. Fragments A, B and D, but not fragment C, were recovered pure by gel chromatography as monitored by polyacrylamide-gel electrophoresis. 3. A complex between fragments C and D was also isolated (mol.wt. 10000) after limited oxidation of flagellin by chloramine-t before digestion by cyanogen bromide. After oxidation essentially only two fragments were released from flagellin by cyanogen bromide: the `C,D' complex and a presumed `AB' fragment. 4. The sum of the amino acid analyses of fragments A and B and the `C,D' complex gave residue values that agreed well with the amino acid composition of native flagellin. 5. Fragments A and D contained tyrosine, and ten of the 11 ∈-N-methyl-lysine residues of the molecule were in fragment A. Reaction with [125I]iodide at small extents of substitution showed that, in flagellin, the tyrosine residue of fragment D was more readily substituted than those of fragment A. By contrast, in polymerized flagellin, the tyrosine residues of fragment A were more readily substituted. 6. Treatment of flagellin with carboxypeptidases A and B revealed the C-terminal sequence -Leu-Leu-Leu-Arg. Arginine and leucine were released by carboxypeptidase from the `C,D' complex but not from fragment D, indicating that fragment C was C-terminal. 7. On the basis of the results from amino acid analysis, carboxypeptidase digestion, N-terminal analysis, iodination studies and polyacrylamide-gel electrophoresis, the sequence of fragments in flagellin was considered to be B–A–D–C; in the polymer, fragment A was exposed. It is suggested that methylation of the lysine residues occurred in the organism after flagellin had polymerized.

Full text

PDF
489

Images in this article

492Fig. 2.
on p.492
495Fig. 4.
on p.495

Selected References

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

  1. ADA G. L., NOSSAL G. J., PYE J., ABBOT A. ANTIGENS IN IMMUNITY. I. PREPARATION AND PROPERTIES OF FLAGELLAR ANTIGENS FROM SALMONELLA ADELAIDE. Aust J Exp Biol Med Sci. 1964 Jun;42:267–282. [PubMed] [Google Scholar]
  2. ADA G. L., NOSSAL G. J., PYE J. ANTIGENS IN IMMUNITY. III. DISTRIBUTION OF IODINATED ANTIGENS FOLLOWING INJECTION INTO RATS VIA THE HIND FOOTPADS. Aust J Exp Biol Med Sci. 1964 Jun;42:295–310. [PubMed] [Google Scholar]
  3. Atassi M. Z., Saplin B. J. Immunochemistry of sperm whale myoglobin. I. The specific interaction of some tryptic peptides and of peptides containing all the reactive regions of the antigen. Biochemistry. 1968 Feb;7(2):688–698. doi: 10.1021/bi00842a026. [DOI] [PubMed] [Google Scholar]
  4. Benoiton L., Deneault J. The hydrolysis of two epsilon-N-methyl-L-lysine derivatives by trypsin. Biochim Biophys Acta. 1966 Mar 7;113(3):613–616. doi: 10.1016/s0926-6593(66)80021-8. [DOI] [PubMed] [Google Scholar]
  5. CRESTFIELD A. M., MOORE S., STEIN W. H. The preparation and enzymatic hydrolysis of reduced and S-carboxymethylated proteins. J Biol Chem. 1963 Feb;238:622–627. [PubMed] [Google Scholar]
  6. Crumpton M. J. An antigenic site of sperm whale myoglobin. Nature. 1967 Jul 1;215(5096):17–20. doi: 10.1038/215017a0. [DOI] [PubMed] [Google Scholar]
  7. DAVIS B. J. DISC ELECTROPHORESIS. II. METHOD AND APPLICATION TO HUMAN SERUM PROTEINS. Ann N Y Acad Sci. 1964 Dec 28;121:404–427. doi: 10.1111/j.1749-6632.1964.tb14213.x. [DOI] [PubMed] [Google Scholar]
  8. Diener E., Armstrong W. D. Induction of antibody formation and tolerance in vitro to a purified protein antigen. Lancet. 1967 Dec 16;2(7529):1281–1285. doi: 10.1016/s0140-6736(67)90394-7. [DOI] [PubMed] [Google Scholar]
  9. GREENWOOD F. C., HUNTER W. M., GLOVER J. S. THE PREPARATION OF I-131-LABELLED HUMAN GROWTH HORMONE OF HIGH SPECIFIC RADIOACTIVITY. Biochem J. 1963 Oct;89:114–123. doi: 10.1042/bj0890114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. HOFMANN T. THE PURIFICATION AND PROPERTIES OF FRAGMENTS OF TRYPSINOGEN OBTAINED BY CYANOGEN BROMIDE CLEAVAGE. Biochemistry. 1964 Mar;3:356–364. doi: 10.1021/bi00891a010. [DOI] [PubMed] [Google Scholar]
  11. Kabat E. A. The nature of an antigenic determinant. J Immunol. 1966 Jul;97(1):1–11. [PubMed] [Google Scholar]
  12. Kim S., Paik W. K. Studies on the origin of epsilon-N-methyl-L-lysine in protein. J Biol Chem. 1965 Dec;240(12):4629–4634. [PubMed] [Google Scholar]
  13. MCDONOUGH M. W. AMINO ACID COMPOSITION OF ANTIGENICALLY DISTINCT SALMONELLA FLAGELLAR PROTEINS. J Mol Biol. 1965 Jun;12:342–355. doi: 10.1016/s0022-2836(65)80258-3. [DOI] [PubMed] [Google Scholar]
  14. McConahey P. J., Dixon F. J. A method of trace iodination of proteins for immunologic studies. Int Arch Allergy Appl Immunol. 1966;29(2):185–189. doi: 10.1159/000229699. [DOI] [PubMed] [Google Scholar]
  15. Neville D. M., Jr Fractionation of cell membrane protein by disc electrophoresis. Biochim Biophys Acta. 1967 Jan 18;133(1):168–170. doi: 10.1016/0005-2795(67)90051-7. [DOI] [PubMed] [Google Scholar]
  16. Parish C. R., Wistar R., Ada G. L. Cleavage of bacterial flagellin with cyanogen bromide. Antigenic properties of the protein fragments. Biochem J. 1969 Jul;113(3):501–506. doi: 10.1042/bj1130501. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. RAY W. J., Jr, KOSHLAND D. E., Jr Comparative structural studies of phosphoglucomutase and chymotrypsin. Brookhaven Symp Biol. 1960 Nov;13:135–150. [PubMed] [Google Scholar]
  18. Reisfeld R. A., Small P. A., Jr Electrophoretic heterogeneity of polypeptide chains of specific antibodies. Science. 1966 May 27;152(3726):1253–1255. doi: 10.1126/science.152.3726.1253. [DOI] [PubMed] [Google Scholar]
  19. STEERS E., Jr, CRAVEN G. R., ANFINSEN C. B., BETHUNE J. L. EVIDENCE FOR NONIDENTICAL CHAINS IN THE BETA-GALACTOSIDASE OF ESCHERICHIA COLI K12. J Biol Chem. 1965 Jun;240:2478–2484. [PubMed] [Google Scholar]
  20. Young J. D., Benjamini E., Shimizu M., Leung C. Y. Immunochemical studies on the tobacco mosaic virus protein. 3. The degradation of an immunologically active tryptic peptide of tobacco mosaic virus protein and the reactivity of the degradation products with antibodies to the whole protein. Biochemistry. 1966 May;5(5):1481–1488. doi: 10.1021/bi00869a006. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES