Separation of different forms of the fourth component of human complement by fast protein liquid chromatography.
Disruption of the thiolester in native C4 yields a 'C4b-like C4' molecule (iC4) that functionally resembles C4b and is therefore probably accompanied by conformational changes in the C4 molecule. In most purified C4 preparations, iC4 and C4b are present to a variable extent. In this study we evaluated the use of fast protein liquid chromatography (FPLC) to resolve and isolate these various forms of C4. C4 was purified from fresh human plasma in a 4-step procedure that included barium citrate adsorption, polyethylene glycol 6000 (PEG) precipitation, Q-Sepharose Fast Flow and mono Q ion exchange chromatography. The final preparation appeared to be homogeneous on SDS-PAGE and under reducing conditions consisted of three bands that corresponded to the intact alpha, beta and gamma chains of C4. In some preparations the alpha' chain of C4b was also observed. On a Mono Q column the purified C4 preparations could be separated into three peaks that by hemolytic assay and SDS-PAGE were characterized as representing native C4, and monomeric and dimeric iC4 (or monomeric and dimeric C4b). Finally, the apparent KA of the various forms of C4 for C4b-binding protein (C4BP) was investigated. The monomeric iC4 and C4b species demonstrated similar C4BP binding affinity with an apparent KA of 5.6-6.4 x 10(8) M-1, whereas their dimeric forms demonstrated a higher affinity for C4BP with an apparent KA: 0.9-2.3 x 10(9) M-1. Binding of native C4 to C4BP was undetectable.
Hessing
M
M
Central Laboratory of The Netherlands Red Cross Transfusion Service, Amsterdam.
Paardekooper
J
J
Hack
C E
CE
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D002853:Chromatography, Liquid; D003176:Complement C3 / Q000378:metabolism; D003181:Complement C4 / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D015935:Complement C4b / Q000032:analysis / Q000302:isolation & purification; D006801:Humans; D016167:Integrin alphaXbeta2; D011817:Rabbits
D002352:Carrier Proteins; D003176:Complement C3; D003181:Complement C4; D016167:Integrin alphaXbeta2; D015935:Complement C4b