Crystallization of human complement component C5.

Human complement component C5 has been crystallized using a low-salt batch technique. The crystals are large hexagonal bi-pyramids often larger than 1.5 mm. Although these crystals were grown in low salt (0.1 M NaCl), they are remarkably stable for at least 2 months at 281 K and they are not dissolved in aqueous buffers containing up to 2 M sodium chloride. The space group is P3121 or P3221, and the cell parameters were determined to be a = 144.9, b = 144.9, c = 243.1 A; alpha = 90 degrees, beta = 90, gamma = 120 degrees. At room temperature and cryo-temperatures the crystals diffract at best to 6 A using rotating-anode X-ray sources. Using synchrotron radiation with cryoprotection using 40%(v/v) PEG 400 the resolution limit can be extended to 3.3 A. In both cases the crystals show significant anisotropy, with relatively weaker reflections at higher resolution in the a*b* plane.

Activation of complement by 405-nm light in serum from porphyria cutanea tarda.

Sera from three patients with porphyria cutanea tarda (PCT) were examined for evidence of complement activation. The irradiation of sera in vitro with 405-nm light resulted in a dose-dependent diminution of total hemolytic complement activity and the hemolytic titers of C1, C4, C2, C3, and C5. Furthermore, such irradiated sera showed immunoelectrophoretical C3 conversion, chemotactic activity for rat polymorphonuclear leukocytes inhibited by incubation with anti-C5 antisera but not anti-C3 antisera, and C5a generation as measured by radioimmunoassay. Factor-B conversion did not occur in such irradiated sera. Using Sephadex G-75 chromatography, the irradiated sera showed a multiphasic elution profile of chemotactic activity similar to that of zymosan-activated serum. The generation of C5a even occurred in factor-B-depleted serum. These studies indicate that the irradiation of PCT serum with 405-nm light induces the activation of the complement system via the classical pathway, resulting in the development of a chemotactic factor.

Complement-derived chemotactic activity is generated in human serum containing uroporphyrin after irradiation with 405 nm light.

Patients with porphyrias have varying degrees of photosensitivity, associated with elevated levels of porphyrins in plasma, erythrocyte, urine and/or feces. To investigate the role of complement in the pathogenesis of cutaneous lesions, varying amounts of uroporphyrin were added to normal human serum (0.1-10 microgram/ml), and the mixtures were then exposed to 405 nm irradiation. Such treatments result in the diminution of total hemolytic complement activity and hemolytic titers of C1, C4, C2, C3, and C5; furthermore, cleavage products of C3 and C5 were detected. Chemotactic activity for human polymorphonuclear leukocytes was generated that was inhibitable by incubation with anti-C5, but not with anti-C3 antisera. No chemotactic activity was generated in Mg++-EGTA treated serum nor in C4-deficient guinea pig serum. These data indicate that irradiation with 405 nm light of normal human serum containing uroporphyrin results in activation of the complement system via the classical pathway, and the generation of complement (C5)-derived chemotactic activity for human polymorphonuclear leukocytes.