PURIFICATION AND CHARACTERIZATION OF A SINGLE CHAIN PRECURSOR C3-PROTEIN (PRO-C3) FROM NORMAL HUMAN-PLASMA

Fresh human plasma was treated with proteinase inhibitors and passed through an immunoadsorbent column of Sepharose anti-C3 [complement component 3] globulin. The insolubilized C3 was eluted with 5 M guanidine and after extensive dialysis was reduced with 0.2 M 2-mercaptoethanol and electrophoresed on SDS[sodium dodecyl sulfate]-polyacrylamide gels. The bulk of the eluted C3 dissociated into 2 major protein bands (MW 125,000 and 75,000) corresponding to the .alpha.- and .beta.-chains of C3. A nonreducible single polypeptide chain (SPC) with a MW value of 197,000 .+-. 2000 similar to the apparent MW of unreduced C3 was consistently present. SPC was purified by elution from SDS (SPC*) and it remained a single polypeptide chain upon re-electrophoresis on SDS gels in the presence of 0.2 M 2-mercaptoethanol. SPC* reacted with antisera to denatured C3, C3.alpha. and C3.beta. chains. Antisera to SPC* also reacted with denatured C3, C3.alpha.- and C3.beta.-chains, revealed a reaction of identity between SPC and C3 and detected partial identity between SPC and C3.alpha.- and C3.beta.-chains. This suggested that SPC and C3 are antigenically related. The amino acid compositions and tryptic peptide maps of SPC and C3 were similar. SPC may represent a single-chain precursor C3 (pro-C3) in plasma that escaped post-synthetic proteolytic cleavage into a 2-subunit chain C3 molecule.