Physicochemical and immunological characterization of the type E botulinum neurotoxin binding protein purified fromClostridium botulinum |
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Authors: | Bal Ram Singh John Foley and Catherine Lafontaine |
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Institution: | (1) Department of Chemistry, University of Massachusetts Dartmouth, 02747 North Dartmouth, Massachusetts;(2) Division of Endocrinology, Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut |
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Abstract: | Type E botulinum neurotoxin is produced byClostridium botulinum along with a neurotoxin binding protein which helps protect the neurotoxin from adversepH, 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% -helix, 50% -sheets, 28% random coils, and 3% -turns. This compared to 22% -helix, 44% -sheets, 34% random coils, and no -turns of the type E botulinum neurotoxin. The complex of the two proteins revealed 25% -helix, 45% -sheets, 27% random coils, and 3% -turns, suggesting a significant alteration at least in the -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 lowpH as indicated by an initial binding rate of 8.4 min–1 atpH 5.7 compared to 4.0 min–1 atpH 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. |
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Keywords: | Botulinum circular dichroism Clostridium ELISA fluorescence interaction neurotoxin binding protein secondary structure tyrosine exposure |
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