Physicochemical and immunological characterization of the type E botulinum neurotoxin binding protein purified from Clostridium botulinum.
Type E botulinum neurotoxin is produced by Clostridium botulinum along with a neurotoxin binding protein which helps protect the neurotoxin from adverse pH, temperature, and proteolytic conditions. The neurotoxin binding protein has been purified as a 118-kDa protein. Secondary structure content of the neurotoxin binding protein as revealed by far-UV circular dichroism spectroscopy was 19% alpha-helix, 50% beta-sheets, 28% random coils, and 3% beta-turns. This compared to 22% alpha-helix, 44% beta-sheets, 34% random coils, and no beta-turns of the type E botulinum neurotoxin. The complex of the two proteins revealed 25% alpha-helix, 45% beta-sheets, 27% random coils, and 3% beta-turns, suggesting a significant alteration at least in the alpha-helical folding of the two proteins upon their interaction. Tyrosine topography is altered considerably (28%) when the neurotoxin and its binding protein are separated, indicating strong interaction between the two proteins. Gel filtration results suggested that type E neurotoxin binding protein clearly complexes with type E neurotoxin. The interaction is favored at low pH as indicated by an initial binding rate of 8.4 min-1 at pH 5.7 compared to 4.0 min-1 at pH 7.5 as determined using a fiber optic-based biosensor. The neurotoxin and its binding protein apparently are of equivalent antigenicity, as both reacted equally on enzyme-linked immunosorbent assay to polyclonal antibodies raised against the toxoid of their complex.
Singh
B R
BR
Department of Chemistry, University of Massachusetts Dartmouth, North Dartmouth 02747, USA.
Foley
J
J
Lafontaine
C
C
D000818:Animals; D001905:Botulinum Toxins / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry* / Q000276:immunology; D006736:Horses; D009498:Neurotoxins / Q000378:metabolism*; D017510:Protein Folding; D017433:Protein Structure, Secondary
D002352:Carrier Proteins; D009498:Neurotoxins; D001905:Botulinum Toxins