Effects of thiolation on the immunoreactivity of the ribosome-inactivating protein gelonin.
Gelonin purified from the seeds of Gelonium multiflorum using cation-exchange and gel-filtration chromatography was characterized for its purity, homogeneity and Mr by reverse-phase h.p.l.c. and SDS/polyacrylamide-gel electrophoresis analysis and judged to be 98% pure. As the cross-linking agent N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) has been used for linking gelonin via its epsilon-NH2 group to its carrier antibodies or hormones for immunotoxin or hormonotoxin respectively, an attempt was made to study the effect of this modification of gelonin on its immunoreactivity. A radioimmunoassay was developed for this purpose. By sequential modification, four categories of amino group modifications on immunoreactivity were observed. Even one or two modifications, representing one-twentieth to one-tenth of available epsilon-NH2 groups in the protein caused about 75% loss in immunoreactivity, with additional reactions contributing to further deteriorations. By using a gelonin radioimmunoassay, the immunoreactivity of gelonin in three hormonotoxins was determined with gelonin and modified gelonin as standards. The gelonin equivalent in our hormonotoxins was in agreement with the values determined by spectrophotometric and gel-electrophoresis methods. As the immunoreactivity of gelonin-SPDP was not further altered after conjugation to its carrier protein ovine lutropin, a specific radioimmunoassay of gelonin could be used to evaluate the molar ratio of the conjugates prepared by using SPDP as cross-linker and gelonin-SPDP as a standard.
Singh
V
V
Reproduction Research Laboratory, Clinical Research Institute of Montreal, Quebec, Canada.
Sairam
M R
MR
D000941:Antigens / Q000276:immunology; D055598:Chemical Phenomena; D002621:Chemistry; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents; D004337:Drug Carriers; D004591:Electrophoresis, Polyacrylamide Gel; D007986:Luteinizing Hormone; D010940:Plant Proteins / Q000276:immunology* / Q000302:isolation & purification; D011863:Radioimmunoassay; D054789:Ribosome Inactivating Proteins, Type 1; D012639:Seeds / Q000032:analysis; D013388:Succinimides; D013438:Sulfhydryl Compounds
D000941:Antigens; D003432:Cross-Linking Reagents; D004337:Drug Carriers; D010940:Plant Proteins; D054789:Ribosome Inactivating Proteins, Type 1; D013388:Succinimides; D013438:Sulfhydryl Compounds; C018151:N-succinimidyl 3-(2-pyridyldithio)propionate; C025674:GEL protein, Gelonium multiflorum; D007986:Luteinizing Hormone