Purification of group C streptococcal extracellular antigens detected with naturally occurring human antibodies: isolation of streptokinase and two previously undescribed antigens.

Twelve antigens were detected in crude group C streptococcal extracellular concentrates, using naturally occurring antibodies in normal human gamma globulin. These group C streptococcal antigens all appeared to be present in crude group A streptococcal extracellular concentrates, although the latter contained additional antigens reactive with the human antibodies. Systematic purification procedures were established for the isolation of the group C streptococcal antigens by a sequence of salting out, hydroxylapatite chromatography, Sephadex G-100 gel filtration, and isoelectric focusing. With such procedures, three of the group C streptococcal antigens were isolated in a relatively pure state. One of the purified antigens was identified as streptokinase on the basis of its fibrinolytic potency, its reaction of identity with two purified streptokinase fractions obtained from other sources, and its high titer in immunodiffusion assays. The most highly purified streptokinase fractions, derived from the 0.1 M sodium phosphate hydroxylapatite eluate, revealed a plasmin-inhibiting effect at high concentrations of streptokinase. This was not seen in the purified streptokinase of equivalent functional and immunological purity that was derived from the 0.2 M sodium phosphate hydroxylapatite peak. Two other streptococcal antigens were also isolated to a high degree during the course of the above study. These were designated antigens X and Y and were found to be unrelated immunologically to each other or to streptokinase. Their isoelectric points were 6.7 and 8.8, respectively, and both were present in group A streptococcal concentrates. Esterase activity was found to be widely distributed in almost all of the fractions obtained in the various purification steps, indicating a high degree of heterogeneity of the streptococcal enzyme. Histochemical staining techniques applied to the immune precipitates formed with human antibodies indicated that none of the antigens detected in crude group C and group A streptococcal concentrates possessed catalase, glucuronidase, glucosaminidase, acid or alkaline phosphatase, arylsulfatase, leucineaminopeptidase, or chymotrypsin enzymatic activities.