Purification of the human complement control protein C3b inactivator

Abstract

An alternative method of isolation from human plasma is described for C3b [b fragment of complement component 3] inactivator, C3bINA, the proteinase that in conjunction with .beta.1H or C4b-binding protein will hydrolyze, respectively, C3b or C4b, the activation products of C3 and C4. The purification is by chromatography of plasma on columns of QAE [quaternary aminoethyl]-Sephadex, wheat-germ agglutinin-Sepharose, hydroxyapatite and Sephacryl S-200. The yield of C3bINA (6 mg from 500 ml plasma) is several-fold higher than in previously described methods. The sensitivity of the assay for C3bINA was increased by including optimal amounts of .beta.1H; .beta.1H was essential for hydrolysis by C3bINA of C3b, whether the C3b was in solution or bound to a cell surface. Native C3 is not hydrolyzed by C3bINA + .beta.1H, but the hemolytically inactive form that appears on prolonged storage at 4.degree. C or on freezing and thawing is hydrolyzed and gives fragments of the .alpha.-chain of 75,000 and 43,000 apparent MW. As the .alpha.''-chain of C3b, which has lost an N-terminal peptide C3a, gives fragments of 67,000 and 43,000 apparent MW when incubated with C3bINA + .beta.1H, the larger fragment is probably N-terminal and the smaller one C-terminal. The pH optimum of C3bINA with soluble substrates is 6.0; no clear classification of the type of proteinase to which this enzyme belongs was obtained.