The membrane attack mechanism of complement. Isolation and subunit composition of the C5b-9 complex

Area of Science:

• Immunology
• Biochemistry
• Complement System

Background:

• The complement system is crucial for innate and adaptive immunity.
• Understanding complement complexes like C5b-9 is vital for immune response research.
• Previous studies have characterized individual complement proteins but not the intact C5b-9 complex structure.

Purpose of the Study:

• To isolate and characterize the C5b-9 complement complex from human serum.
• To determine the protein composition and subunit stoichiometry of the C5b-9 complex.
• To identify novel components within the C5b-9 complex.

Main Methods:

• Isolation using molecular sieve chromatography and preparative electrophoresis.
• Purification via ultracentrifugation and lipoprotein removal.
• Analysis by Ouchterlony double diffusion and SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

Main Results:

• Successfully isolated a homogeneous C5b-9 complex.
• Confirmed the presence of C5b, C6, C7, C8, and C9 via Ouchterlony analysis.
• SDS-PAGE revealed seven protein bands, identifying C5b, C6, C7, C9, and C8 subunits.
• Estimated molar ratios of 1:1:1:1:3 for C5b:C6:C7:C8:C9.
• Identified an unknown protein (Band 5) with a molar ratio of 3.

Conclusions:

• The C5b-9 complex is composed of multiple complement proteins with specific stoichiometry.
• The identified unknown protein may play a significant role in complement function.
• Further research is needed to elucidate the structure and function of the unidentified component.