PURIFICATION AND CHARACTERIZATION OF A PROTEASE FROM CLOSTRIDIUM-BOTULINUM TYPE-A THAT NICKS SINGLE-CHAIN TYPE-A BOTULINUM NEUROTOXIN INTO THE DI-CHAIN FORM

A protease that nicks the ~150-kilodalton (kDa) single-chain type A botulinum neurotoxin into the ~150-kDa di-chain form in vitro was isolated from Clostridium botulinum type A (Hall strain) cultures. The di-chain neurotoxin generated in vitro is composed of an ~50-kDa light chain and an ~-100-kDa heavy chain which are disulfide linked and is industinguishable from the di-chain neurotoxin that forms in vivo and is routinely isolated (M.L. Dekleva and B.R. DasGupta, Biochem. Biophys. Res. Commun. 162:767-772, 1989). This enzyme was purified >1,000-fold by ammonium sulfate precipitation, QAE-Sephadex Q-50, Sephadex G-100, and CM-Sephadex C-50 chromatography steps with the synthetic substrate N-benzoyl-DL-arginine-p-nitroanilide. The ~62-kDa amidase (protease) is a complex of 15.5 and 48-kDa polypeptides (determined by polyacrylamide gel electrophoresis) that could not be separated without sodium dodecyl sulfate. The enzyme has an isoelectric point of pH 5.73, a pH optimum of 6.2 to 6.4, an absolute requirement for a thiol-reducing agent as well as a divalent metallic cation (probably Ca2+) for activity, and a temperature optimum of 70°C. Tests with several synthetic substrates indicated the high specificity of the enzyme for arginyl amide bonds.