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Inactivation of C3 by enzymatic cleavage, nucleophilic addition, or slow freezing and thawing resulted in the acquisition of similar end-state conformations as judged by near-UV circular dichroism. Although inactivation by the two nonenzymatic processes involves no peptide bond scission, the inactivated C3 resembled C3b in that it possessed a free sulfhydryl group not present in the native protein and an increased surface hydrophobicity as evidenced by enhanced binding of the fluorophore 8-anilino-1-naphthalensulfonate (ANS). The C3b-like functional properties of modified C3 [Pangburn, M. K., & Muller-Eberhard, H. J. (1980) J. Exp. Med. 152, 1102-1114] may thus be understood in terms of the similarity of its conformation to that of C3b. The rate of the conformational change following proteolytic cleavage was fast and appeared to be limited by the rate of the enzymatic reaction. In contrast, the rate of conformational change following addition of methylamine was slow and rate limited by the conformational rearrangement itself, not by the chemical modification. A kinetic analysis of the changes in circular dichroism and ANS fluorescence enhancement suggested that the nucleophilic addition was spectroscopically undetectable and was followed by a minimally biphasic, spectroscopically demonstrable conformational rearrangement. The appearance of C3b-like functional activity in nucleophile-modified C3 largely parallels the time course of the spectroscopically detectable conformational change but is distinctly slower than the rate at which hemolytic activity is lost. While fully transconformed methylamine-inactivated C3 can bind factor B and is susceptible to cleavage by C3b inactivator and its cofactor beta 1H, this cleavage occurs at a substantially slower rate than the equivalent process in C3b. The implications of these findings in terms of the mechanism through which the alterative pathway of complement is initiated are discussed.

A lambda light chain, isolated from an immunoglobulin G molecule, was found to reversibly precipitate at low temperatures. This cryoprecipitation was a function of pH, ionic strength, protein concentration, and time as well as temperature. The lambda chain underwent a cooperative conformational change as the temperature was lowered from 26 to 0 degrees C as judged by ultraviolet difference spectroscopy and circular dichroism. Normal lambda chains showed no conformational change. By difference spectroscopy it was possible to calculate the equilibrium constant governing the conformational change. The change was strongly exothermic (delta H approximately -80 kcal mol-1) and accompanied by a large decrease in entropy (delta S approximately -280 eu). The midpoint of the transition was dependent on the initial protein concentration, suggesting that only the noncovalent dimer of the lambda chain exhibited the conformational change. The existence of a monomer-dimer eqiulibrium (KA approximately 4 X 10(5) M-1) was confirmed by sedimentation velocity. No conformational change was observed by circular dichroism at concentrations where greater than 95% of lambda chain was in the form of a monomer. Although high ionic strength inhibited cryoprecipitation, it had no effect on the conformational change. Stabilization of the dimer by forming an interchain disulfide bond between two monomers abolished both the conformational change and cryoprecipitation. A fragment corresponding to the constant region was isolated from both peptic and tryptic digests of the lambda chain. This fragment neither cryoprecipitated nor showed temperature dependence conformational changes. It proved impossible to isolate a fragment corresponding to the variable region. Both qualitative and quantitative models are presented to account for the behavior of the lambda chain at low temperatures.