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The Protein Journal

, Volume 32, Issue 2, pp 106–117 | Cite as

Molecular Composition and Extinction Coefficient of Native Botulinum Neurotoxin Complex Produced by Clostridium botulinum Hall A Strain

  • Anne-Marie Bryant
  • Jenny Davis
  • Shuowei Cai
  • Bal Ram Singh
Article

Abstract

Seven distinct strains of Clostridium botulinum (type A to G) each produce a stable complex of botulinum neurotoxin (BoNT) along with neurotoxin-associated proteins (NAPs). Type A botulinum neurotoxin (BoNT/A) is produced with a group of NAPs and is commercially available for the treatment of numerous neuromuscular disorders and cosmetic purposes. Previous studies have indicated that BoNT/A complex composition is specific to the strain, the method of growth and the method of purification; consequently, any variation in composition of NAPs could have significant implications to the effectiveness of BoNT based therapeutics. In this study, a standard analytical technique using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and densitometry analysis was developed to accurately analyze BoNT/A complex from C. botulinum type A Hall strain. Using 3 batches of BoNT/A complex the molar ratio was determined as neurotoxin binding protein (NBP, 124 kDa), heavy chain (HC, 90 kDa), light chain (LC, 53 kDa), NAP-53 (50 kDa), NAP-33 (36 kDa), NAP-22 (24 kDa), NAP-17 (17 kDa) 1:1:1:2:3:2:2. With Bradford, Lowry, bicinchoninic acid (BCA) and spectroscopic protein estimation methods, the extinction coefficient of BoNT/A complex was determined as 1.54 ± 0.26 (mg/mL)−1cm−1. These findings of a reproducible BoNT/A complex composition will aid in understanding the molecular structure and function of BoNT/A and NAPs.

Keywords

Botulinum Clostridium Complex Composition Extinction coefficient Molecular ratio 

Abbreviations

BoNT

Botulinum neurotoxin

BoNT/A

Botulinum neurotoxin type A

NAPs

Neurotoxin-associated proteins

NBP

Neurotoxin-binding protein

HC

Heavy chain

LC

Light chain

TC

Toxin complex

M-TC

Medium toxin complex

L-TC

Large toxin complex

LL-TC

Extra-large toxin complex

є

Extinction coefficient

SDS–PAGE

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

BCA

Bicinchoninic acid

BSA

Bovine serum albumin

TBST

Tris buffered saline containing 0.05 % Tween-20

BoNT/D

Botulinum neurotoxin type D

Notes

Acknowledgments

We would like to thank Paul Lindo and Stephen Riding for BoNT/A complex purification. National Institutes of Health Grant 1U01A1078070-02 supported this work.

Conflict of interest

The authors declare that there are no conflicts of interest.

Ethical standards

The authors declare that the experiments comply with the current laws of the United States of America.

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Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Anne-Marie Bryant
    • 1
  • Jenny Davis
    • 1
  • Shuowei Cai
    • 1
  • Bal Ram Singh
    • 1
  1. 1.Botulinum Research Center and Department of Chemistry and BiochemistryUniversity of Massachusetts DartmouthNorth DartmouthUSA

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