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The covalent-binding reaction of complement component C3

R B Sim, T M Twose, D S Paterson

    The Biochemical Journal
    |January 1, 1981
    PubMed
    Summary
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    The complement protein C3 forms a reactive intermediate that covalently binds surfaces. This study quantifies C3b binding kinetics and identifies nucleophiles that label the C3d active site.

    Area of Science:

    • Biochemistry
    • Immunology
    • Protein Chemistry

    Background:

    • The complement system protein C3 is crucial in immune responses.
    • Activated C3 forms a transient C3b intermediate capable of covalent surface binding.

    Purpose of the Study:

    • To characterize the covalent binding reaction of complement protein C3.
    • To determine the kinetics and efficiency of C3b formation and binding.
    • To identify the active site of C3 using nucleophilic labeling.

    Main Methods:

    • Utilized Sepharose-trypsin as a proteolytic activator and binding surface for C3.
    • Quantified C3b binding saturation and calculated the reactive intermediate's lifetime.
    • Investigated the effect of pH and ionic strength on C3 binding efficiency.

    Related Experiment Videos

  • Employed radioactive nucleophiles to identify the C3d fragment as the binding site.

    • Main Results:

    • C3 binding to Sepharose-trypsin is saturable, with a calculated intermediate lifetime of ~60 microseconds.
    • Optimal binding efficiency (>30%) occurs at physiological pH and ionic strength.
    • Binding efficiency decreases with increasing pH and high ionic strength.
    • Radioactive nucleophiles covalently incorporated into the C3d fragment, acting as active-site labels.

    Conclusions:

    • The reactive intermediate of C3 has a short but significant lifetime.
    • Covalent C3b binding is sensitive to environmental conditions like pH and ionic strength.
    • Nucleophilic labeling confirms the C3d fragment as the active site for covalent bond formation.