Purification and Characterization of two Molecular Forms of Bovine Complement Factor H

Abstract

Complement factor H was highly purified from bovine serum using a combination of tyrosine-O-sulfate (TyrS)-Affi-Gel 10 affinity chromatography, DEAE-BioGel A ion-exchange chromatography, and hydroxylapatite chromatography. The purified bovine factor H migrated as doublet protein bands with apparent molecular weights of ca. 160,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. The two forms of the purified factor H were investigated with respect to the sensitivity to limited trypsin digestion. The high-molecular weight form was cleaved into two fragments with apparent molecular masses of, respectively, 45 kD and 125 kD. The low-molecular weight form was cleaved in a different manner to generate three major fragments with molecular masses of, respectively, 25 kD, 45 kD and 100 kD. Limited V8 protease mapping of the two forms of factor H yielded similar, although not identical, peptide band patterns. N-terminal amino acid sequencing revealed that the 13 amino acid residues of the two forms identified are identical, suggesting that they may in fact be isoforms of the same protein. Purified factor H appeared to bind agarose-bonded TyrS or heparin through its anion-binding domain. Furthermore, the binding to TyrS or heparin bonded to agarose was inhibited by the presence of free heparin or dextran sulfate.