Botulinum Research Center and Department of Chemistry and Biochemistry, University of Massachusetts Dartmouth, North Dartmouth, Massachusetts 02747, USA.
Anal Chem. 2012 Dec 18;84(24):10549-53. doi: 10.1021/ac302997n. Epub 2012 Dec 5.
Botulinum neurotoxins (BoNTs), which are highly toxic proteins responsible for botulism, are produced by different strains of Clostridium botulinum. These various strains of bacteria produce seven distinct serotypes, labeled A-G. Once inside cells, the zinc-dependent proteolytic light chain (LC) degrades specific proteins involved in acetylcholine release at neuromuscular junctions causing flaccid paralysis, specifically synaptosomal-associated protein 25 (SNAP-25) for botulinum neurotoxin type A (BoNT/A). BoNT endopeptidase assays using short substrate homologues have been widely used and developed because of their ease of synthesis, detection limits, and cost. SNAPtide, a 13-amino acid fluorescence resonance energy transfer (FRET) peptide, was used in this study as a SNAP-25 homologue for the endopeptidase kinetics study of BoNT/A LC. SNAPtide uses a fluorescein isothiocyanate/4-((4-(dimethylamino)phenyl)azo) benzoic acid (FITC/DABCYL) FRET pair to produce a signal upon substrate cleavage. Signal quenching can become an issue after cleavage since quencher molecules can quench cleaved fluorophore molecules in close proximity, reducing the apparent signal. This reduction in apparent signal provides an inherent error as SNAPtide concentrations are increased. In this study, fluorescence internal quenching (FIQ) correction factors were derived using an unquenched SNAPtide peptide to quantify the signal quenching over a range of SNAPtide concentrations and temperatures. The BoNT/A LC endopeptidase kinetics at the optimally active temperature (37 °C) using SNAPtide were studied and used to demonstrate the FIQ correction factors in this study. The FIQ correction factors developed provide a convenient method to allow for improved accuracy in determining and comparing BoNT/A LC activity and kinetics using SNAPtide over a broad range of concentrations and temperatures.
肉毒杆菌神经毒素（BoNTs）是导致肉毒中毒的高度毒性蛋白，由不同株的肉毒梭菌产生。这些不同的细菌株产生七种不同的血清型，分别标记为 A-G。一旦进入细胞，锌依赖性蛋白水解轻链（LC）会降解参与神经肌肉接头乙酰胆碱释放的特定蛋白，导致弛缓性瘫痪，具体来说是突触相关蛋白 25（SNAP-25），用于肉毒神经毒素 A 型（BoNT/A）。由于其易于合成、检测限和成本，使用和开发短底物类似物的 BoNT 内切酶测定已被广泛应用。在这项研究中，使用了一种 13 个氨基酸的荧光共振能量转移（FRET）肽 SNAPtide 作为 SNAP-25 类似物，用于 BoNT/A LC 的内切酶动力学研究。SNAPtide 使用异硫氰酸荧光素/4-((4-(二甲基氨基)苯基)偶氮)苯甲酸（FITC/DABCYL）FRET 对来产生信号，当底物被切割时产生信号。由于淬灭剂分子可以淬灭附近的切割荧光团分子，从而降低表观信号，因此在切割后，信号猝灭可能成为一个问题。由于 SNAPtide 浓度的增加，这种表观信号的减少会导致固有误差。在这项研究中，使用未猝灭的 SNAPtide 肽衍生出荧光内部猝灭（FIQ）校正因子，以定量在一系列 SNAPtide 浓度和温度下的信号猝灭。在最佳活性温度（37°C）下，使用 SNAPtide 研究了 BoNT/A LC 内切酶动力学，并在本研究中证明了 FIQ 校正因子的应用。开发的 FIQ 校正因子提供了一种方便的方法，可以在广泛的浓度和温度范围内，提高使用 SNAPtide 确定和比较 BoNT/A LC 活性和动力学的准确性。
