Two dimensional crystals of botulinum toxin, serotype B

Botulinum toxin is a powerful, protein neurotoxin produced by Clostridium botulinum which exerts its toxic action by inhibiting the release of acetyl choline. There are several immunologically distinguishable types of botulinum toxin and most of these have been shown to bind the ganglioside GTlb. The ganglioside binding property of these neurotoxins has allowed us to prepare two dimensional crystals of serotypes A, B, and E. We report here our preliminary observations of two dimensional crystals of serotype B.Type B botulinum toxin was purified by methods reported by DasGupta and Woody. The two dimensional crystals were prepared by slightly modified procedures used previously to prepare similar crystals of tetanus and cholera toxins. Purified toxin was dialyzed into citric acid-sodium phosphate buffer at pH 4.0 to 6.5 and ionic strength of about 0.04. Dialyzed toxin was placed into the wells of microtiter dishes in 20 μl volumes at a concentration of fifty to one hundred μgm per ml. The toxin solutions were then layered with one to two μl of a solution of one to two mg per ml of egg lecithin (Sigma Cat. No. P- 2772) in chloroform containing five to ten percent by weight of the ganglioside GT1B (Supelco Cat. No. 4-6035). The microtiter dishes were then placed in the cold and crystallization was allowed to proceed for one to four days at 4°C. The crystals were then picked up on carbon coated electron microscope grids, negatively stained with one to two percent uranium acetate and examined in the electron microscope.