The fifth component of complement (C5): purification without activation

In the course of our studies on the structural change of C5 by acidification (U. Rother et al., 1978), we found that the C5 preparations purified according to published methods contained more or less activated C56. When added to sensitive target cells (guinea pig or chicken erythrocytes), C5 mediated lysis by C7-C9 without the addition of C6 or any activation procedure. Generation of C56 was probably due to drastic changes in the physicochemical environment during purification. Such changes like high or low pH or high ionic strength were shown to cause activation. A method for purification of C5 is described in which polyethyleneglycol (PEG) or (NH4)2SO4 precipitation, as well as low or high pH, was avoided. As a last step, traces of C6 were removed by affinity chromatography. The resulting preparation was free of C56. Activation by acidification was not possible without the addition of C6. The total recovery of C5 was 12% with almost no loss of specific activity.