The weight-average molecular weight of Cl (r) over bar, an activated s
erine protease subcomponent of complement Cl, was measured in the pres
ence of widely varying concentrations of Ca2+ and the other serine pro
tease subcomponent, Cl (s) over bar, by utilizing the technique of tra
cer sedimentation equilibrium. A quantitative model for heteroassociat
ion between the two subcomponents, which takes into account the previo
usly observed Ca2+-dependent self-association of Cl (s) over bar s, wa
s fit to the combined data at each Ca2+ concentration. The results ind
icate that Cl (r) over bar, which exists as a dimer under all of the c
onditions explored in this work, can bind up to two molecules of Cl (s
) over bar at both low and high Ca2+ concentrations, but the associati
on constant for binding a single molecule of Cl (s) over bar to dimeri
c Cl (r) over bar is estimated to increase on the order of a 1000-fold
as [Ca2+] increases from 1 nM to 1.0 mM. Heteroassociation of Cl (r)
over bar and Cl (s) over bar is favored over self-association of Cl (s
) over bar at all conditions. The results clearly indicate the necessi
ty of taking into account a multiplicity of states of association when
attempting to understand the equilibrium average properties of a mixt
ure of the two subcomponents and their binding Clq in solution.