Human C4-binding protein. I. Isolation and characterization

Abstract

C4[4th component of complement]-binding protein (C4-bp), a new component of the complement system, was isolated from human plasma by precipitation with polyethyleneglycol, followed by chromatography on ion exchangers. C4-bp was identified on sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) by 2 independent criteria: its ability to bind to C4b and immunoprecipitation with a monospecific antiserum. Purified C4-bp is a 10.7 S glycoprotein. It consists of several disulfide bonded subunits of MW 70,000 daltons. Under nonreducing conditions, its MW was estimated on SDS-PAGE as 540-590,000 daltons. C4-bp moves as a slow .beta.-globulin at pH 8.6 in the absence of free divalent cations, but when the buffers contain Ca2+-lactate, C4-bp is a .gamma.-globulin. Purified C4-bp binds to purified C4b. The reaction proceeds in the presence or absence of divalent cations and is not inhibited by diisopropylfluorophosphate. The C4b/C4-bp complexes have sedimentation coefficients between 15 and 17 S on sucrose gradient ultracentrifugation, and can be readily identified by crossed immunoelectrophoresis (CIE). The complexes move faster toward the anode than either protein. C4-bp is multivalent. Saturation is reached at molecular rations of C4b/C4-bp of between 4 and 5. The interaction C4b and C4-bp may complicate the electrophoretic patterns of these proteins in normal human serum, if the complement system is activated before or duing in normal human serum, if the complement system is activated before or during the run. However, in EDTA-plasma, native C4 and C4-bp do not form stable complexes and can be identified in separate peaks after CIE.