Acquisition of C 3 b - like Activities by Spontaneous Hydrolysis of the Putative Thioester in Native C 3 ' Be MICHAEL K . PANGBURN , ROBERT D . SCHREIBER , AND HANS

We present evidence suggesting that nonenzymatic, spontaneous hydrolysis of a thioester bond in native C3 constitutes the initial event in the activation of the alternative pathway. It has been known since the original description of C3 that the hemolytic activity of the isolated protein decays spontaneously in aqueous solution at a slow rate (1). This decay was greatly accelerated by chaotropic agents (2). It has also been known that treatment of C3 with simple amines such as hydrazine abolished its hemolytic activity (3). Recently, evidence has accumulated that strongly supports the existence of an intramolecular thioester bond in native C3 (4-8). Modification of the thioester by incorporation of 1 mol of methylamine destroyed the potential of C3 to bind to biological targets of complement, induced conformational changes in the molecule (9) and led to the expression of functional binding sites for complement proteins resembling those of enzymatically generated C3b (4, 10). Methylamine-modified C3 [C3(CHsNH2)] 1 was shown (4) to bind Factor B and properdin and was susceptible to cleavage and inactivation by Factors H and I. The present study was performed to investigate the spontaneous decay of the binding potential of native C3 and to determine whether hydrolysis of the thioester bond in C3 generates, without any further chemical or enzymatic modification, C3blike functional properties. Because chaotropic agents facilitate access of water to relatively concealed groups by perturbing the tertiary structure of proteins, these agents were used to accelerate the spontaneous decay of C3. Chaotrope-treated C3 that has acquired C3b-like functions on the basis of this treatment will be tentatively designated C3(H20). This denotation proposes that this form of C3 contains, instead of the thioester bond, a free sulfhydryl and carboxyl group.