Factor D of the alternative pathway of bovine complement: isolation and characterization

Factor D of the bovine alternative complement pathway has been purified by chromatography on CM-Sephadex C-50, Sephacryl S-200 and hydroxylapatite. The isolated factor D (0.25 mg from 1 litre of bovine serum) had an apparent molecular weight of 27,500 and a pI of 7.2. In whole bovine serum the pI of factor D was also 7.2. The isolated protein caused the Mg++-dependent cleavage of bovine factor B in the presence of cobra venom factor (CVF) to generate a haemolytically active C3-convertase as shown by SDS-polyacrylamide gel electrophoresis and haemolytic diffusion plate assays. Bovine factor D and human factor D were interchangeable in restoring the alternative pathway haemolytic activities of both bovine RD and human RD (factor D deficient sera). The haemolytic activity of bovine serum factor D was completely inhibited by 20 mM diisopropylfluorophosphate (DFP) but only 25% inhibited by 1 mM DFP. Serum heated at 56 degrees C for 10 min completely lost factor D activity but purified factor D was relatively more heat stable.