Purification of the human complement control protein C3b inactivator.
An alternative method of isolation from human plasma is described for C3b inactivator, C3bINA, the proteinase that in conjunction with either beta 1H or C4b-binding protein will hydrolyse respectively C3b or C4b, the activation products of the third, C3 and fourth, C4, components of complement. The purification is by chromatography of plasma on columns of QAE-Sephadex, wheat-germ agglutinin-Sepharose, hydroxyapatite and Sephacryl S-200. The yield of C3bINA (6 mg from 500ml of plasma) is severalfold higher than in previously described methods. The sensitivity of the assay for C3bINA has been increased by including optimal amounts of beta 1H, and it was observed that 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 hydrolysed by C3bINA + beta 1H, but the haemolytically inactive form that appears on prolonged storage at 4 degrees C or on freezing and thawing is hydrolysed and gives fragments of the alpha-chain of 75000 and 43000 apparent mol.wt. As the alpha'-chain of C3b, which has lost an N-terminal peptide C3a, gives fragments of 67000 and 43000 apparent mol.wt. when incubated with C3bINA + beta 1H, this suggests that the larger fragment is N-terminal and the smaller one C-terminal. The pH optimum of C3bINA with soluble substrates is 6.0, but no clear classification of the type of proteinase to which this enzyme belongs has been obtained.
D002850:Chromatography, Gel; D003176:Complement C3; D003179:Complement C3b; D003180:Complement C3b Inactivator Proteins / Q000097:blood / Q000302:isolation & purification*; D017242:Complement Factor H; D004591:Electrophoresis, Polyacrylamide Gel; D006461:Hemolysis; D006801:Humans; D006863:Hydrogen-Ion Concentration; D011480:Protease Inhibitors
C493495:CFH protein, human; D003176:Complement C3; D003180:Complement C3b Inactivator Proteins; D011480:Protease Inhibitors; D003179:Complement C3b; D017242:Complement Factor H