Botulinum Research Center and Department of Chemistry and Biochemistry, University of Massachusetts Dartmouth, 285 Old Westport Road, North Dartmouth, MA 02747, USA.
Protein J. 2013 Feb;32(2):106-17. doi: 10.1007/s10930-013-9465-6.
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)(-1)cm(-1). These findings of a reproducible BoNT/A complex composition will aid in understanding the molecular structure and function of BoNT/A and NAPs.
七种不同的肉毒梭菌菌株（A 到 G 型）各自产生稳定的肉毒神经毒素（BoNT）复合物，以及神经毒素相关蛋白（NAPs）。A型肉毒神经毒素（BoNT/A）与一组 NAPs 一起产生，可用于治疗多种神经肌肉疾病和美容用途。先前的研究表明，BoNT/A 复合物的组成特定于菌株、生长方法和纯化方法；因此，NAPs 组成的任何变化都可能对基于 BoNT 的治疗的有效性产生重大影响。在这项研究中，开发了一种使用十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）和密度计分析的标准分析技术，以准确分析来自 A 型 Hall 株肉毒梭菌的 BoNT/A 复合物。使用 3 批 BoNT/A 复合物，确定了摩尔比为神经毒素结合蛋白（NBP，124 kDa）、重链（HC，90 kDa）、轻链（LC，53 kDa）、NAP-53（50 kDa）、NAP-33（36 kDa）、NAP-22（24 kDa）、NAP-17（17 kDa），为 1:1:1:2:3:2:2。通过 Bradford、Lowry、双缩脲（BCA）和分光光度法蛋白质估计方法，确定 BoNT/A 复合物的消光系数为 1.54±0.26（mg/mL）（-1）cm（-1）。这些重现性 BoNT/A 复合物组成的发现将有助于理解 BoNT/A 和 NAPs 的分子结构和功能。
