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Related Experiment Videos

The ninth component of human complement: purification and physicochemical characterization.

G Biesecker, H J Müller-Eberhard

    Journal of Immunology (Baltimore, Md. : 1950)
    |March 1, 1980
    PubMed
    Summary

    Researchers isolated the human complement protein C9, a glycoprotein crucial for forming the membrane attack complex. This purified C9 is essential for understanding complement-mediated cell lysis and immune responses.

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    Area of Science:

    • Immunology
    • Biochemistry
    • Molecular Biology

    Background:

    • The human complement system is a critical part of innate immunity.
    • The terminal pathway of complement activation involves the formation of the membrane attack complex (MAC).
    • Complement protein 9 (C9) is the final component of the MAC, responsible for pore formation in target cell membranes.

    Purpose of the Study:

    • To describe a procedure for the isolation of human complement protein C9.
    • To characterize the biochemical and biophysical properties of purified C9.
    • To investigate the role of C9 in complement-mediated cell lysis.

    Main Methods:

    • Protein purification techniques.
    • Electrophoresis (gel and immunochemical).

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  • Mass spectrometry for molecular weight determination.
  • Automated Edman degradation for N-terminal analysis.
  • Radioimmunoassay or ELISA for serum concentration determination.
  • Complement-mediated cell lysis assays.
  • Electron microscopy for ultrastructural analysis.

    • Main Results:

    • A procedure for isolating pure human C9 was established.
    • Purified C9 is an alpha-globulin with a molecular weight of 71,000 Da, consisting of a single polypeptide chain.
    • C9 is a glycoprotein containing approximately 7.8% carbohydrate, including glucosamine, neutral hexose, and sialic acid.
    • The N-terminus of C9 is blocked.
    • Normal human serum contains C9 at a concentration of 58 ± 8 μg/ml.
    • Depletion of C9 from serum did not affect overall hemolytic activity (CH50) but prevented the formation of typical membrane attack complex lesions on lysed erythrocytes.

    Conclusions:

    • A reliable method for C9 isolation and characterization was developed.
    • C9 is a key component for the formation of the membrane attack complex's ultrastructural lesions.
    • While C9 is essential for MAC lesion formation, its absence does not abolish overall complement-mediated hemolysis, suggesting alternative pathways or roles in lysis.