Isolation and characterization of the eighth component of the bovine complement system

Objective-To isolate and characterize the eighth component of the complemen t system (C8) in cattle. Sample Population-Fresh plasma obtained from beef cattle. Procedures-Plasma samples were fractionated, using sequential precipitation and ion-exchange and gel-filtration chromatography, to yield C8. The prote in was identified throughout the procedure on the basis of its hemolytic fu nction. Electrophoresis in polyacrylamide gels was used to determine molecu lar weight and composition of polypeptide chains. Reconstitution of classic al and alternative complement pathways was used to characterize the hemolyt ic function of bovine C8. Results-The bovine C8 protein consisted of a disulfide-bonded alpha-gamma h eterodimer that was noncovalently associated with a beta chain. Apparent mo lecular weight of the alpha, beta, and gamma chains under reducing conditio ns were 66, 61, and 23 kd, respectively. In the classical pathway of activa tion, bovine C8 and the ninth component of the complement system (C9) had s pecies incompatibility with human C8 and C9 on sheep erythrocyte target cel ls. Conclusions-A simple 4-step fractionation procedure provided good yield of bovine C8 from plasma. The isolated protein was structurally comparable to C8 from other species. Purified bovine C8 may be useful in functional hemol ytic assays to investigate the roles of complement-mediated lysis in the pa thogenesis of inflammatory diseases and the killing of susceptible microorg anisms.