Characterization of group A streptococcal R-28 antigen purified by hydroxyapatite column chromatography.
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.
D000596:Amino Acids / Q000032:analysis; D000645:Ammonium Sulfate; D000818:Animals; D000942:Antigens, Bacterial / Q000032:analysis* / Q000302:isolation & purification; D011232:Chemical Precipitation; D002845:Chromatography; D004591:Electrophoresis, Polyacrylamide Gel; D006882:Hydroxyapatites; D011817:Rabbits; D013297:Streptococcus pyogenes / Q000276:immunology*
D000596:Amino Acids; D000942:Antigens, Bacterial; D006882:Hydroxyapatites; D000645:Ammonium Sulfate