Subunit stoichiometry of the Clostridium botulinum type A neurotoxin complex determined using denaturing capillary electrophoresis.
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.
Lietzow
Michael A
MA
Biopharmaceuticals Department, Allergan, Inc, 2525 Dupont Drive, Irvine, CA 92612, USA. lietzow_michael@allergan.com
Gielow
Elizabeth T
ET
Le
Denise
D
Zhang
Jifeng
J
Verhagen
Marc F
MF
D019274:Botulinum Toxins, Type A / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D019075:Electrophoresis, Capillary / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D008970:Molecular Weight; D009498:Neurotoxins / Q000737:chemistry*; D011487:Protein Conformation; D021122:Protein Subunits; D012542:Scattering, Radiation
D009498:Neurotoxins; D021122:Protein Subunits; D019274:Botulinum Toxins, Type A