Affinity chromatographic purification and characterization of two iodinated tetanus toxin fractions exhibiting different binding properties.
Highly purified iodinated tetanus toxin preparations separate on ganglioside affinity columns into two distinct (A and B) fractions representing about 20% and 75% of the iodinated toxin, respectively. Fraction A, eluted by 1% NaCl, migrates like native tetanus toxin (150,000 mol. wt) on SDS polyacrylamide gel electrophoresis. It forms an aggregate of molecular weight approximately 360,000 on Sephacryl S-300 gel permeation chromatography in the presence of detergent and contains two isoforms on preparative chromatofocusing. Fraction A binds poorly to neurons in tissue culture or to synaptosomal membrane preparations. It retains, however, its antigenicity and biotoxicity. Fraction B, eluted by 6% NaCl, binds effectively to gangliosides and also to neurons or synaptosome preparations. It has a similar molecular weight and chain composition to the native toxin and displays two isoforms, precipitable during chromatofocusing. Fraction B possesses similar binding, immunological and toxic properties to the original iodinated tetanus toxin. Following excessive iodination (4-6 mCi/mg protein), toxicity can be remarkably reduced. Unlabeled toxin shows a similar chromatographic pattern to the iodinated toxin on affinity columns, suggesting that a large portion (30% by protein and 55% by toxicity) of the toxin has a poor affinity for gangliosides. The molecular pharmacokinetics of tetanus toxin with respect to affinity toward ganglioside-dependent and ganglioside-independent receptors needs re-evaluation.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification; D001345:Autoradiography; D002540:Cerebral Cortex / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004912:Erythrocytes / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007525:Isoelectric Focusing; D051379:Mice; D008807:Mice, Inbred BALB C; D008970:Molecular Weight; D051381:Rats; D013574:Synaptosomes / Q000378:metabolism; D013744:Tetanus Toxin / Q000302:isolation & purification* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D013744:Tetanus Toxin