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. 1975 Oct;12(4):901–909. doi: 10.1128/iai.12.4.901-909.1975

Characterization of group A streptococcal R-28 antigen purified by hydroxyapatite column chromatography.

R H Johnson
PMCID: PMC415372  PMID: 1104480

Abstract

Purified R-28 antigen from an M-protein-poor, R-antigen-rich strain of group A Streptococcus was prepared by sequential treatment of an acid extract of whole cells with ammonium sulfate fractionation and hydroxylapatite (HA) column chromatography. Purified R-28 antigen was eluted only with 0.10 M sodium phosphate, pH 6.7. Findings on quantitative amino acid composition, polyacrylamide gel electrophoresis pattern, and HA column elution pattern were similar but not identical to those previously reported for streptococcal M-proteins. Rabbits immunized with either HA-purified R-28 antigen or heat-killed cells developed two pepsin-sensitive, trypsin-resistant immunodiffusion lines of identity against HA-purified R-28 antigen but failed to form bactericidal antibody. One of these two lines formed a line of identity with R-28 antigen prepared by trypsinization of whole cells. The other line remained undefined, although it appeared not to be either streptococcal group A carbohydrate, M-protein, T-antigen, polyglycerophosphate, E4 antigen, or M-associated protein; by enzymatic criteria it is an R-antigen. Polyacrylamide gel electrophoresis of HA-purified R-28 antigen revealed multiple serologically active charge and size isomers. These findings suggest possible structural similarities between group A streptococcal M-proteins and R-antigens and also indicate that the same purification techniques may be utilized to study these protein antigens if the proper strain of Streptococcus is chosen.

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Selected References

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

  1. 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]
  2. Erwa H. H. Studies on two methods for extraction of streptococcal T antigens. J Hyg (Lond) 1973 Mar;71(1):131–138. doi: 10.1017/s0022172400046295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Fox E. N. M proteins of group A streptococci. Bacteriol Rev. 1974 Mar;38(1):57–86. doi: 10.1128/br.38.1.57-86.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Fox E. N., Wittner M. K. New observations on the structure and antigenicity of the M proteins of the group A streptococcus. Immunochemistry. 1969 Jan;6(1):11–24. doi: 10.1016/0019-2791(69)90174-8. [DOI] [PubMed] [Google Scholar]
  5. Fox E. N., Wittner M. K. The multiple molecular structure of the M proteins of group A streptococci. Proc Natl Acad Sci U S A. 1965 Oct;54(4):1118–1125. doi: 10.1073/pnas.54.4.1118. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Hedrick J. L., Smith A. J. Size and charge isomer separation and estimation of molecular weights of proteins by disc gel electrophoresis. Arch Biochem Biophys. 1968 Jul;126(1):155–164. doi: 10.1016/0003-9861(68)90569-9. [DOI] [PubMed] [Google Scholar]
  7. Jackson R. W., Moskowitz M. Nature of a red cell sensitizing substance from streptococci. J Bacteriol. 1966 Jun;91(6):2205–2209. doi: 10.1128/jb.91.6.2205-2209.1966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Johnson R. H., Vosti K. L. Purification of two fragments of M protein from a strain of group A, type 12 streptococcus. J Immunol. 1968 Sep;101(3):381–391. [PubMed] [Google Scholar]
  9. LANCEFIELD R. C. Differentiation of group A streptococci with a common R antigen into three serological types, with special reference to the bactericidal test. J Exp Med. 1957 Oct 1;106(4):525–544. doi: 10.1084/jem.106.4.525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. LANCEFIELD R. C. Occurrence of R antigen specific for Group A type 3 streptococci. J Exp Med. 1958 Sep 1;108(3):329–341. doi: 10.1084/jem.108.3.329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. LANCEFIELD R. C., PERLMANN G. E. Preparation and properties of a protein (R antigen) occurring in streptococci of group A, type 28 and in certain streptococci of other serological groups. J Exp Med. 1952 Jul;96(1):83–97. doi: 10.1084/jem.96.1.83. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  13. MOODY M. D., PADULA J., LIZANA D., HALL C. T. EPIDEMIOLOGIC CHARACTERIZATION OF GROUP A STREPTOCOCCI BY T-AGGLUTINATION AND M-PRECIPITATION TESTS IN THE PUBLIC HEALTH LABORATORY. Health Lab Sci. 1965 Jul;2:149–162. [PubMed] [Google Scholar]
  14. McLEAN S. J. Identification of strains of Streptococcus pyogenes of Types 5, 11, 12, 27 and 44 by the precipitin test for the T antigen. J Gen Microbiol. 1953 Aug;9(1):110–118. doi: 10.1099/00221287-9-1-110. [DOI] [PubMed] [Google Scholar]
  15. Moskowitz M. Separation and properties of a red cell sensitizing substance from streptococci. J Bacteriol. 1966 Jun;91(6):2200–2204. doi: 10.1128/jb.91.6.2200-2204.1966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Myoda T. T., Wiley G. G., Bruno P. N. Cross-reactions among group A streptococci. IV. Extraction, separation and purification of two protective antigens of type G 1 cocci. J Immunol. 1973 Jul;111(1):249–259. [PubMed] [Google Scholar]
  17. OUCHTERLONY O. Antigen-antibody reactions in gels. IV. Types of reactions in coordinated systems of diffusion. Acta Pathol Microbiol Scand. 1953;32(2):230–240. [PubMed] [Google Scholar]
  18. PAKULA R. Extraction of the T antigen of Streptococcus pyogenes. J Gen Microbiol. 1951 Oct;5(4):640–647. doi: 10.1099/00221287-5-4-640. [DOI] [PubMed] [Google Scholar]
  19. Straus D. C., Mehta A., Lange C. F. Simplified method for the purification of group A streptococcal M-proteins: solution of the multiple banding problem. Appl Microbiol. 1974 Jan;27(1):28–37. doi: 10.1128/am.27.1.28-37.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Vosti K. L., Johnson R. H., Dillon M. F. Further characterization of purified fractions of M protein from a strain of group A, type 12 Streptococcus. J Immunol. 1971 Jul;107(1):104–114. [PubMed] [Google Scholar]
  21. WILSON A. T., WILEY G. G. THE CELLULAR ANTIGENS OF GROUP A STREPTOCOCCI; IMMUNOELECTROPHORETIC STUDIES OF THE C, M, T, PGP, E4, F, AND E ANTIGENS OF SEROTYPE 17 STREPTOCOCCI. J Exp Med. 1963 Oct 1;118:527–556. doi: 10.1084/jem.118.4.527. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Widdowson J. P., Maxted W. R., Pinney A. M. An M-associated protein antigen (MAP) of group A streptococci. J Hyg (Lond) 1971 Dec;69(4):553–564. doi: 10.1017/s0022172400021823. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Wilkinson H. W. Comparison of streptococcal R antigens. Appl Microbiol. 1972 Oct;24(4):669–670. doi: 10.1128/am.24.4.669-670.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Zumwalt R. W., Kuo K., Gehrke C. W. A nanogram and picogram method for amino acid analysis by gas-liquid chromatography. J Chromatogr. 1971 May 6;57(2):193–208. doi: 10.1016/0021-9673(71)80033-x. [DOI] [PubMed] [Google Scholar]

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