Purification and characterization of three toxins and two agglutinins from Abrus precatorius seed by using lactamyl-Sepharose affinity chromatography.
Three toxins, abrin-I, -II, and -III, and two agglutinins, APA-I and -II, were purified from the seeds of Abrus precatorius by lactamyl-Sepharose affinity chromatography followed by gel filtration and DEAE-Sephacel column chromatography. Abrin-I did not bind on DEAE-Sephacel column chromatography and the bound abrin-II, abrin-III, APA-I, and APA-II were eluted with a sodium acetate gradient. The identity of each protein was established by sodium dodecylsulfate-polyacrylamide gel electrophoresis and isoelectric focusing. The relative molecular weights are abrin-I, 64,000; abrin-II and abrin-III, 63,000 each: APA-I, 130,000; and APA-II, 128,000. Isoelectric focusing revealed microheterogeneity due to the presence of isoforms in each protein. Toxicity and binding studies further confirmed the differences among the lectins. The time course of inhibition of protein synthesis in thymocytes by the toxins showed lag times of 78, 61, and 72 min with Ki's of 0.55, 0.99, and 0.74 ms-1 at a 0.63 nM concentration of each of abrin-I, -II, and -III, respectively. A Scatchard plot obtained from the equilibrium measurement for the lectins binding to lactamyl-Sepharose beads showed nonlinearity, indicating a cooperative mode of binding which was not observed for APA-I binding to Sepharose 4B beads. Further, by the criterion of the isoelectric focusing profile, it was shown that the least toxic abrin-I and the highly toxic abrin-II isolated by lactamyl-Sepharose chromatography were not retained on a low-affinity Sepharose 4B matrix, which signifies the necessity of using a high-affinity matrix for the purification of the lectins.
Hegde
R
R
Department of Biochemistry, Indian Institute of Science, Bangalore.
Maiti
T K
TK
Podder
S K
SK
D000036:Abrin / Q000302:isolation & purification; D000818:Animals; D050260:Carbohydrate Metabolism; D002846:Chromatography, Affinity / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D006384:Hemagglutination / Q000187:drug effects; D007785:Lactose / Q000378:metabolism*; D037102:Lectins / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D007928:Lethal Dose 50; D051379:Mice; D037121:Plant Lectins; D012639:Seeds; D012685:Sepharose / Q000378:metabolism; D012967:Sodium Dodecyl Sulfate; D014118:Toxins, Biological / Q000302:isolation & purification* / Q000378:metabolism / Q000633:toxicity
D037102:Lectins; D037121:Plant Lectins; D014118:Toxins, Biological; D000036:Abrin; D012967:Sodium Dodecyl Sulfate; D012685:Sepharose; D007785:Lactose