The membrane attack mechanism of complement: the three polypeptide chain structure of the eigth component (C8)

Area of Science:

• Biochemistry
• Immunology
• Proteomics

Background:

• Human complement component 8 (C8) is crucial for the membrane attack complex formation.
• Understanding C8 structure and function requires pure protein for detailed analysis.

Purpose of the Study:

• To develop a method for purifying milligram quantities of human C8.
• To characterize the subunit composition of purified human C8.

Main Methods:

• Ammonium sulfate precipitation (37.5-50% saturation) of outdated human serum.
• Ion exchange column chromatography using CM-32 cellulose and QAE-Sephadex.
• SDS-polyacrylamide gel electrophoresis (SDS-PAGE) for subunit analysis.

Main Results:

• Achieved milligram-scale purification of human C8 with a 1,700-fold average purification.
• Yield of C8 activity ranged from 2-9%.
• SDS-PAGE of reduced C8 identified three equimolar polypeptide chains: alpha (77 kDa), beta (63 kDa), and gamma (13.7 kDa).
• Denaturation revealed two noncovalently linked subunits: alpha-gamma (99 kDa) and beta (75 kDa).

Conclusions:

• A robust protocol for human C8 purification was established.
• The study elucidated the multi-chain polypeptide structure of human C8.
• Detailed characterization of C8 subunits provides a foundation for further functional studies.