Schmidt J J, Stafford R G, Millard C B
Department of Cell Biology and Biochemistry, Toxinology and Aerobiology Division, U.S. Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 21702-5011, USA.
Anal Biochem. 2001 Sep 1;296(1):130-7. doi: 10.1006/abio.2001.5236.
Botulinum neurotoxins (BoNT) are zinc metalloproteases that cleave and inactivate cellular proteins essential for neurotransmitter release. Because the paralytic effect of BoNT is a consequence of its enzymatic activity, selective inhibitors may be useful as drugs or as tools for further research. To expedite inhibitor discovery, we developed high-throughput, solid-phase protease activity assays for four of the seven BoNT serotypes: A, B, D, and F. Each assay consisted of a cleavable oligopeptide, based on the natural substrate sequence, labeled with fluorescein and covalently attached to maleimide-activated multiwell plates. Solutions of holotoxin or nontoxic catalytic domain of BoNT were incubated in substrate-coated wells, with or without test compounds, followed by transfer and assay of solubilized product in a multiwell fluorometer. Routine toxin concentrations ranged from 10 to 100 ng/ml, but concentrations as low as 2 ng/ml gave reproducible signals. The fluorescence assays were selective, gave very low background readings, and were stable upon prolonged storage. Using the nontoxic catalytic domain of BoNT A, we determined the relative inhibitory potencies of a family of structurally related pseudotripeptide compounds. Unlike previous methods, our assays did not employ antibodies or reverse-phase extraction steps, only well-to-well transfers, and were easily adapted to a high-throughput automated environment.
肉毒杆菌神经毒素（BoNT）是锌金属蛋白酶，可切割并使神经递质释放所必需的细胞蛋白失活。由于BoNT的麻痹作用是其酶活性的结果，选择性抑制剂可作为药物或进一步研究的工具。为了加快抑制剂的发现，我们针对七种BoNT血清型中的四种：A、B、D和F，开发了高通量固相蛋白酶活性测定法。每种测定法都包含一种基于天然底物序列的可切割寡肽，用荧光素标记并共价连接到马来酰亚胺活化的多孔板上。将BoNT的全毒素或无毒催化结构域溶液在包被有底物的孔中孵育，有或没有测试化合物，然后转移并在多孔荧光计中测定溶解产物。常规毒素浓度范围为10至100 ng/ml，但低至2 ng/ml的浓度也能给出可重复的信号。荧光测定法具有选择性，背景读数非常低，并且在长时间储存后仍很稳定。使用BoNT A的无毒催化结构域，我们确定了一系列结构相关的假三肽化合物的相对抑制效力。与以前的方法不同，我们的测定法不使用抗体或反相萃取步骤，只进行孔间转移，并且很容易适应高通量自动化环境。
