Related Experiment Videos
Separation of different forms of the fourth component of human complement by fast protein liquid chromatography
M Hessing1, J Paardekooper, C E Hack
1Central Laboratory of The Netherlands Red Cross Transfusion Service, Amsterdam.
Insights
Researchers developed a method using fast protein liquid chromatography (FPLC) to separate different forms of complement C4, including native C4, monomeric and dimeric C4b-like C4 (iC4), and C4b. This technique revealed distinct binding affinities of these C4 variants to C4b-binding protein (C4BP).
Area of Science:
- Immunology
- Biochemistry
- Complement System
Background:
- Native complement C4 can be converted to a C4b-like molecule (iC4) through thiolester disruption, exhibiting functional similarities to C4b.
- Purified C4 preparations often contain variable amounts of iC4 and C4b, necessitating methods for their resolution.
- Understanding the different forms of C4 and their interactions is crucial for elucidating complement system pathways.
Purpose of the Study:
- To evaluate the efficacy of fast protein liquid chromatography (FPLC) for resolving and isolating native C4, iC4, and C4b.
- To characterize the separated C4 forms using hemolytic assays and SDS-PAGE.
- To investigate the binding affinities of different C4 forms to C4b-binding protein (C4BP).
Main Methods:
- Four-step purification of C4 from human plasma involving adsorption, PEG precipitation, and ion-exchange chromatography (Q-Sepharose and Mono Q).
- Separation of purified C4 into distinct peaks using Mono Q FPLC.
- Characterization of separated peaks via hemolytic assays and SDS-PAGE.
- Determination of apparent binding affinities (KA) for C4b-binding protein (C4BP) using various C4 forms.
Main Results:
- FPLC successfully separated purified C4 into three distinct peaks identified as native C4, monomeric iC4/C4b, and dimeric iC4/C4b.
- Monomeric iC4 and C4b exhibited similar binding affinities to C4BP (apparent KA: 5.6-6.4 x 10^8 M^-1).
- Dimeric iC4 and C4b displayed significantly higher binding affinities to C4BP (apparent KA: 0.9-2.3 x 10^9 M^-1).
- Binding of native C4 to C4BP was undetectable.
Conclusions:
- FPLC is an effective method for resolving and isolating different functional forms of complement C4.
- Monomeric and dimeric forms of iC4/C4b display differential binding affinities to C4BP, with dimeric forms showing higher affinity.
- These findings provide insights into the heterogeneity of C4 in plasma and its interactions within the complement cascade.
Abstract:
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.