Biotinylation of human C3.
Purified human C3 was biotinylated using the biotinyl-N-hydroxysuccinimide imidoester (BNHS). Depending on the input of BNHS, from three to six molecules of biotin were incorporated per C3 molecule. The biotinyl-C3 retained over 90% of its specific hemolytic activity and when bound to sheep erythrocytes maintained its ability to adhere to human C3b receptors. These functions could be blocked by avidin. The biotinyl-C3 was fragmented normally to C3c and C3d in human serum and adsorption with avidin-Sepharose indicated that biotin moities were present in both fragments. Fluorescein-conjugated avidin reacted well with cell-bound biotinyl-C3b and was useful for quantitating C3 fixation by flow cytometry. Ferritin-conjugated avidin was used as a marker to characterize the distribution of biotinyl-C3b on erythrocytes by electron microscopy. These results suggest that biotinyl-C3 and avidin derivatives may be very useful tools for studies of many of the biological functions of C3.
D001360:Avidin / Q000494:pharmacology; D001710:Biotin / Q000031:analogs & derivatives; D003176:Complement C3 / Q000276:immunology; D005434:Flow Cytometry; D006461:Hemolysis / Q000187:drug effects; D006801:Humans; D007122:Immunoelectrophoresis; D008854:Microscopy, Electron; D011487:Protein Conformation; D013388:Succinimides
D003176:Complement C3; D013388:Succinimides; C033788:biotinyl N-hydroxysuccinimide ester; D001360:Avidin; D001710:Biotin