Abstract We studied the early events in activation of the classical complement (C) pathway by treating normal human serum (NHS) with different C activators and measuring the following parameters as a function of time: C1 activation, C2 consumption, C4 consumption, and C1 inactivation. C1 activation and inactivatidn were quantitated with SDS-PAGE radioassays, whereas C2 and C4 consumption were measured by hemolytic titration. In undiluted NHS at 37°C, C1 activation as well as C2 and C4 consumption were turned off within 3 min after the addition of limiting quantities of activator. The binding of C1-inactivator (C1-ln) to C1 was so rapid that no free C1 was detected at any time during the reaction. Direct binding studies with purified proteins revealed that, under physiologic conditions, 50% of the C1 bound to C1-In within 13 sec and 90% within 55 sec of activation. Thus, the activation of C1 is the rate limiting step for this 3-min reaction sequence. C1 turned over a surprisingly low number of C2 and C4 molecules in NHS. This was due to the fact that C1 was so rapidly inactivated by C1-ln. An average of only 4 C2 molecules and 35 C4 molecules Were activated per C1 molecule when soluble agglgG, tetanus-anti-tetanus precipitate, EAIgG, or EAIgM were added to NHS. The nonimmune activator LPS R595 differed from these immune activators in that it induced the activation of 5 times less C2 per C1. The Michaelis-Menten constant (0.96 × 10−6 M) and turnover number (303 C4/C1/min) were calculated for the activation of purified C4 by purified C1. These values are consistent with the observed turnover of C4 in NHS when the short half-life of C1 is taken into account. The early classical pathway was reconstituted from purified C1q, C1r, C1s, C1-ln, C2, and C4, all at physiologic concentrations. When treated with agglgG, this purified component mixture and NHS were indistinguishable with respect to consumption of C2 and C4. Thus, C1-In is the only serum factor involved in the control of C1 activities. Our results together demonstrate that the control protein C1-In is extraordinarily efficient.

Ziccardi, R. J. (1981). Activation of the early components of the classical complement pathway under physiologic conditions. The Journal of Immunology, 126(5), 1769–1773.