Separation and characterization of a 14,000-dalton cyanogen bromide-generated peptide from a 185,000-dalton streptococcal antigen

Abstract

The cell surface streptococcal antigen (SA) (185,000 molecular weight [185K SA]) was isolated from Streptococcus mutans and digested with cyanogen bromide. Three major products with molecular weights of 100,000, 50,000, and 14,000 appeared within 1 h of digestion. Time course studies of digestion by polyacrylamide gel electrophoresis showed maximal intensity of the 14K band after 8 h. However, other bands appeared as well, notably 70K and 20K bands. Several bands were eluted from the gels, and their antigenicity was studied. They reacted with antisera to the native 185K SA I/II, as well as with those to the SA I and SA II antigens, though antibody binding by radioimmunoassay was significantly lower than that with the native SA. The 14K SA was identified on Western blots (immunoblots) with anti-SA I/II, I, and II antisera. The digested SAs were then tested for their immunogenicity by injecting CBA mice with the separated SA mixed in complete Freund adjuvant. Whereas the unseparated cyanogen bromide-treated SA and separated 70K and 20K SAs were immunogenic, the 14K SA failed to elicit serum antibodies. Further investigation of the 14K SA revealed that although it is apparently not immunogenic, it can induce a primary antibody response in mice when followed by the native 185K SA and a secondary response when mice are immunized first with the 185K SA.