The complement system is one of the defense mechanisms of the body. Most complement proteins are present in blood in precursor form; C3, C4, and C5 are among these. The action of all three is under extremely specific control. Precise and unique conformation of each is required for its physiological function. Among the probable factors contributing to the stability of functional forms of these molecules are the disulfide-binding patterns and, in C3 and C4, the presence of the thiolester at the labile binding site. The loss of ability of C3 or C4 to mediate hemolysis is accompanied by the appearance of an SH group from splitting of the thiol ester, and by conformational changes. All three parts of this proposal are intimately related to the structural integrity and functionality of the three complement proteins. 1) A method for absolute determination of hemolytically active molecules in C3 and C4 samples will be developed. It will serve in standardization of other functional methods, and will also be applied to determination of C3(n) and C4(n) in plasma samples. 2) Hemolytically active C3(n) and various forms of spontaneously inactivated C3(i) will be separated and their properties investigated. The final procedure for isolation of C3(n), both on macro- and micro-scale, will be applied with appropriate modifications to the isolation of C4(n). 3) Half-cystinyl residues within each of the proteins will be localized, and their disulfide bonding patterns wil be established. The results of the proposed studies should contribute to a better understanding of molecular properties of C3, C4, and C5, possibly providing further support for suspected homology among them, and should provide a background to further investigations of the overall structure of these proteins, which is necessary for their interactions and physiological functions. Such a knowledge will assist in future clinical intervention in disease states involving the complement.
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