SOLUBILIZATION OF C3-FRAGMENTS DEPOSITED ON CROSS-LINKED DEXTRAN GEL BEADS

Complement activation links C3 fragments to objects by powerful, possibly even covalent bonds. Therefore, it has not been possible to analyze the structure and function of bound C3 fragments except under denaturing conditions. We have succeeded in solubilizing C3 fragments by dissolving the object to which they are bound. Incubation of Sephadex G75 beads with normal human serum in physiologic veronal-buffered saline Mg EGTA, pH 7.4, results in deposition of C3 on the surface of the beads by the alternative complement pathway, as evidenced by its occurrence in C2-deficient serum and in the absence of Ca2+. Washing of serum-treated beads with 0.15 and 2 M NaCl removed all other proteins except for traces of IgG and albumin. The C3 deposited was biologically active, because with this deposition the beads acquired the capacity to activate glucose metabolism by human polymorphonuclear leukocytes. C3 fragments solubilized by treatment of C3-coated G75 beads with Dextranase could be analyzed under nondenaturing conditions by electrophoresis, and for biological activity. Kinetic analysis revealed an initial appearance on the beads of a 175,000-dalton C3 fragment that is similar to C3b by immunochemical and functional criteria. Further incubation resulted in appearance of other fragments of 173,000 and 140,000 daltons, probably representing C3bi and C3c. C3d was not detected. © 1979.