Summary
Human C4b-binding protein (C4BP) is a multimeric regulatory component of the complement
system that circulates in plasma either as a free protein or in a noncovalent complex
with the vitamin K-dependent protein S. The major form of C4BP is composed of seven
identical α-chains (70 kDa) and one β-chain (45 kDa). C4BP was purified from human
plasma after barium citrate adsorption using anti-C4BP monoclonal antibody affinity
chromatography. C4BP-high and low Mr forms were both obtained from the barium citrate precipitate and the supernatant.
C4BP-high and low forms from the barium citrate precipitate were separated by sodium
dodecylsulfate polyacrylamide slab gel electrophoresis and extracted with Triton X-100.
Both forms contained the β-chain as was demonstrated on sodium dodecylsulfate polyacrylamide
slab gel electrophoresis under reduced conditions after silverstaining and with Western-blotting
using monoclonal antibodies specific for the β-chain. The C4BP-high and low forms
demonstrated similar protein S binding affinity (KA: 3.18 × 108 and 3.21 × 108 M-1, respectively) in a C4BP-protein S binding assay and a protein S ligand blot using
a peroxidase-conjugated monoclonal anti-protein S antibody. The barium citrate supernatant
contained two forms of C4BP-high and one form of C4BP-low. One form of C4BP-high did
contain the β-chain and was capable of protein S binding (KA: 4.35 × 108 M-1). The two other forms of C4BP lacked the β-chain and were unable to bind protein
S.
