C1s Bound in Macromolecular C1 Exists in Equilibrium with C1s Free in Serum

We have established the existence of an equilibrium between free C1s and C1s bound in macromolecular C1 in human serum. Untreated human sera were incubated for 35 hours at 4°C with ¹²⁵I-C1s to allow the exchange between free and bound C1s to reach equilibrium. The C1 complex thus labeled was separated from most of the serum proteins and from excess ¹²⁵I-C1s by centrifugation in linear 10–30% sucrose gadients. ¹²⁵I-C1s became incorporated into macromolecular C1, as demonstrated by specific cleavage of all the labeled C1s to C1s̄ after incubation with known activators of the classical pathway of complement. Thus, C1 fractions containing ¹²⁵I-C1s can be used directly and conveniently to assay C1 activation.

Incubating for more than 35 hours at 4°C neither increased nor decreased the amount of ¹²⁵I-C1s in serum C1. Furthermore, excess ¹²⁵I-C1s remained in the free protein pool of samples incubated more than 35 hours and entered C1 upon incubation with additional fresh serum.