Molecular Composition and Extinction Coefficient of Native Botulinum Neurotoxin Complex Produced by Clostridium botulinum Hall A Strain
Molecular Composition and Extinction Coefficient of Native Botulinum Neurotoxin Complex Produced by Clostridium botulinum Hall A Strain
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DOI：
10.1007/s10930-013-9465-6
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发表时间：
2013-02-01
期刊：
影响因子：
3
通讯作者：
Singh, Bal Ram
中科院分区：
文献类型：
--
作者：
Bryant, Anne-Marie;Davis, Jenny;Singh, Bal Ram
肉毒梭菌的七种不同菌株（A型至G型）各自产生肉毒神经毒素（BoNT）沿着神经毒素相关蛋白（NAP）的稳定复合物。A型肉毒杆菌神经毒素（BoNT/A）由一组NAP产生，并且可商购获得，用于治疗许多神经肌肉疾病和美容目的。先前的研究已经表明，BoNT/A复合物组合物对菌株、生长方法和纯化方法是特异性的;因此，NAP组合物的任何变化都可能对基于BoNT的治疗剂的有效性具有显著影响。本研究建立了一种标准的十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）和光密度分析技术，用于准确分析C. A型肉毒杆菌霍尔菌株。使用3批BoNT/A复合物，将摩尔比确定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）1：1：1：2：3：2：2。用Bradford，Lowry，bicinchoninic acid（BCA）和光谱蛋白质估计方法，测定BoNT/A复合物的消光系数为1.54 +/- 0.26（mg/mL）（-1）cm（-1）。这些可重现的BoNT/A复合物组合物的发现将有助于理解BoNT/A和NAP的分子结构和功能。
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.