Modification of the binding site(s) of lectins by an affinity column carrying an activated galactose-terminated ligand.
An affinity column approach is described, aimed at the modification of the galactose binding site(s) of ricin in an effort to block the binding of ricin to cells. The affinity column was prepared by linking N-(2'-mercaptoethyl)lactamine to pyridyldithio-activated polyacrylamide heads. The linker between the ligand and the solid support thus contained a disulfide bond and an unmodified terminal galactose moiety. The amino group of the ligand was allowed to react with the bifunctional cross-linking reagent 2,4-dichloro-6-methoxytriazine. The lectin was then allowed to bind to the galactose functions on the activated column at pH 7.0, prior to raising the pH to 8.6 to initiate the cross-linking reaction between the ligand and the lectin. Lectin that was not covalently linked to the functionalized galactose residues on the column was eluted with galactose or lactose. Finally, the covalent ligand-lectin complexes were released from the solid support by reducing the disulfide bond between the ligand and the support. The affinity column was used in this way to modify the galactose binding site(s) of ricin. Upon release from the affinity column, blocked ricin was purified from unmodified ricin by affinity chromatography on columns of immobilized asialofetuin (a ligand to which ricin binds very tightly). The sulfhydryl group formed by cleavage of the ligand-ricin complex from the column was labeled with [3H]-N-ethylmaleimide to provide evidence that one blocking ligand was linked per ricin molecule. The blocked ricin and a conjugate of the blocked ricin with the monoclonal antibody J5 were toxic for cultures of Namalwa cells in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)
Moroney
S E
SE
Division of Tumor Immunology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115.
D'Alarcao
L J
LJ
Goldmacher
V S
VS
Lambert
J M
JM
Blättler
W A
WA
D000596:Amino Acids / Q000032:analysis; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002846:Chromatography, Affinity / Q000379:methods; D004187:Disaccharides / Q000138:chemical synthesis; D005033:Ethylmaleimide / Q000378:metabolism; D005690:Galactose / Q000378:metabolism*; D007202:Indicators and Reagents; D007700:Kinetics; D008024:Ligands; D011485:Protein Binding; D012276:Ricin / Q000378:metabolism* / Q000633:toxicity; D013438:Sulfhydryl Compounds / Q000138:chemical synthesis
D000596:Amino Acids; D004187:Disaccharides; D007202:Indicators and Reagents; D008024:Ligands; D013438:Sulfhydryl Compounds; C055355:N-(2'-mercaptoethyl)lactamine; D012276:Ricin; D005033:Ethylmaleimide; D005690:Galactose