Purification and characterization of rabbit factor IX and its existence as a two-chain factor IX alpha in circulating plasma.
The long term objective of this study is to immunodeplete rabbits of factor IX as a means of developing a rabbit model for hemophilia B for use in studies of tissue factor-dependent blood coagulation. As a first step, we have purified rabbit factor IX by basically two different methods: (1) conventional chromatography utilizing DEAE-Sephadex and heparin-agarose column chromatography (2) immunoaffinity chromatography on monoclonal anti-rabbit factor IX IgG column. Purified rabbit factor IX migrated as a single band with an apparent molecular mass of 76 kD on nonreduced SDS-PAGE. On reduced SDS-PAGE the majority of factor IX migrated as a two-chain molecule (molecular masses 51 and 28 kD) and a faint band corresponding to 78 kD. We have shown that the purification of rabbit factor IX as a two-chain molecule is not due to the partial proteolysis of factor IX during its purification from commercially obtained rabbit plasma. Analysis of 3H-labelled rabbit factor IXa on SDS-PAGE revealed that, in contrast to human factor IXa, carbohydrate was found associated with the heavy chain of activated factor IX (H beta) after release of the activation peptide. Further understanding of the molecular properties of rabbit factor IX and the generation of neutralizing monoclonal and polyclonal antibodies will facilitate the development of a rabbit model for hemophilia B.
Rao
L V
LV
Department of Medicine, University of California, San Diego 92093.
Hoang
A D
AD
D000595:Amino Acid Sequence; D000818:Animals; D005164:Factor IX / Q000737:chemistry / Q000302:isolation & purification*; D015949:Factor IXa / Q000737:chemistry / Q000302:isolation & purification*; D006801:Humans; D015151:Immunoblotting; D007163:Immunosorbent Techniques; D008969:Molecular Sequence Data; D011817:Rabbits; D012689:Sequence Homology, Nucleic Acid
D005164:Factor IX; D015949:Factor IXa