Nucleation-controlled polymerization of human monoclonal immunoglobulin G cryoglobulins.

The kinetics of the polymerization of human monoclonal cryoimmunoglobulins at low temperature was investigated in temperature jump experiments by monitoring the changes in turbidity resulting from the scattering of incident light by the polymers. Above a critical concentration between 2 and 3 mg/ml, depending on the ionic strength, the kinetics were characterized by a concentration-dependent lag phase and initial rate of self-assembly. Under equilibrium conditions which favored polymerization, the only stable intermediate detected by analytical ultracentrifugation was the dimer. Although purified monomers were unable to self-associate at 4 degrees C, addition of trace amounts of autologous dimers promoted polymerization. The apparent rate of polymerization was shown to be slow (k = 4.7 X 10(-4) M-1 s-1), and the process was governed by an equilibrium constant of 4.6 X 10(4) M-1. The initial rate of self-assembly was proportional to the product of the monomer concentration and the concentration of promoter (i.e. dimer). The rate of depolymerization was three orders of magnitude greater than the rate of polymerization and was proportional to the concentration of polymers present. These results suggest that the polymerization of monoclonal cryoimmunoglobulins is a nucleation-controlled process in which dimerization is the rate-limiting step. Kinetic studies on the polymerization of Fab and F(ab')2 fragments from cryoimmunoglobulins and a comparison of cryogel ultrastructure by electron microscopy suggested that the interaction site between monomers is located in the Fab region. Since the polymerization of monomers was only induced by autologous dimers and not dimers from other cryoimmunoglobulins, it was concluded that the hypervariable regions play a specific role in the condensation reaction. The fact that one cryoimmunoglobulin has a well defined antibody activity against streptolysin O argued against a low temperature-induced auto-anti-idiotype mechanism. Reduction of the interchain disulfide bonds of the Fab fragments abolished their ability to polymerize, probably by inducing a conformational change a considerable distance away in the variable domains of the molecules.