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Subunit Stoichiometry of the Clostridium botulinum Type A Neurotoxin Complex Determined Using Denaturing Capillary Electrophoresis

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Abstract

A denaturing capillary electrophoresis method was developed to evaluate the subunit stoichiometry of the Clostridium botulinum type A neurotoxin complex. The results indicate that the neurotoxin complex contains single copies of the 150 kDa neurotoxin and the non-toxic non-hemagglutinating subunits as well as 5–6 HA17, 4–5 HA23, 3–4 HA48, and 8–9 HA34 subunits. The calculated molecular mass for a complex with this stoichiometry is between 880 and 1,000 kDa. The molecular mass of the intact complex was determined using size-exclusion HPLC (SE-HPLC) and SE-HPLC in conjunction with multi-angle laser light scattering detection. Based on a comparison to a mixture of standard proteins, SE-HPLC analysis yielded a molecular mass of 880 kDa while light scattering analysis indicated a weight average molecular mass of 925 ± 45 kDa. The close agreement between the molecular mass values determined by the three approaches supports the subunit stoichiometry proposed for the C. botulinum type A neurotoxin complex.

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Abbreviations

CE:

Capillary electrophoresis

MALLS:

Multi angle laser light scattering

SE-HPLC:

Size exclusion high performance liquid chromatography

SDS-PAGE:

Sodium dodecyl sulphate polyacrylamide gel electrophoresis

NTNH:

Non-toxic non-hemagglutinating protein

HA:

Hemagglutinin

NAP:

Neurotoxin associated protein

BoNT:

Clostridium botulinum neurotoxin

BoNTA:

Clostridium botulinum type A neurotoxin

Mr:

Molecular mass

RSD:

Relative standard deviation

BSA:

Bovine serum albumin

CA:

Carbonic anhydrase

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Authors and Affiliations

  1. Biopharmaceuticals Department, Allergan, Inc, 2525 Dupont Drive, Irvine, CA, 92612, USA

    Michael A. Lietzow, Elizabeth T. Gielow, Denise Le, Jifeng Zhang & Marc F. Verhagen

Authors
  1. Michael A. Lietzow
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  2. Elizabeth T. Gielow
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  3. Denise Le
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  4. Jifeng Zhang
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  5. Marc F. Verhagen
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Correspondence to Marc F. Verhagen.

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Lietzow, M.A., Gielow, E.T., Le, D. et al. Subunit Stoichiometry of the Clostridium botulinum Type A Neurotoxin Complex Determined Using Denaturing Capillary Electrophoresis. Protein J 27, 420–425 (2008). https://doi.org/10.1007/s10930-008-9151-2

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  • DOI: https://doi.org/10.1007/s10930-008-9151-2

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