کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
1215703 966981 2006 7 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Capillary electrophoresis laser-induced fluorescence for screening combinatorial peptide libraries in assays of botulinum neurotoxin A
موضوعات مرتبط
مهندسی و علوم پایه شیمی شیمی آنالیزی یا شیمی تجزیه
پیش نمایش صفحه اول مقاله
Capillary electrophoresis laser-induced fluorescence for screening combinatorial peptide libraries in assays of botulinum neurotoxin A
چکیده انگلیسی

Botulinum neurotoxin serotype A (BoNT/A) is a proteolytic enzyme that induces muscle paralysis. It is a cause of food poisoning, a potential bioterrorist threat and, in low doses an emerging pharmaceutical product. No effective treatment is currently available for BoNT intoxication. Previously we developed a BoNT/A light chain enzyme assay using a peptide substrate based on the SNAP-25 protein target, with HPLC separation and UV detection of assay products, and applied the method to screen combinatorial peptide libraries for inhibitory activity to BoNT/A. We now report on development of a capillary electrophoresis laser-induced fluorescence (CE-LIF) method for measuring BoNT/A activity. The enzyme assay products were labeled with CBQCA dye followed by CE separation on a bare fused silica column in a HEPES-based buffer and LIF detection. All assay products were separated in CE within 8 min compared to incomplete separation of assay products within 1 h by HPLC. The labeled products showed linear dependence of intensity versus concentration, and quantitative mole-fraction assignments. We used the CE-LIF method to screen combinatorial peptide libraries for potential modulating effects on BoNT/A peptidase activity. With some of the libraries, peptides co-migrated with assay products and interfered with quantitation. In such cases, interference was reduced by substituting sodium dodecyl sulfate (SDS) for Tween-20 in the running buffer. Separation in the capillaries then occurred by micellar electrokinetic chromatography (MEKC). The CE-LIF method is quick and lends itself to high-throughput or microfluidic formats.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Chromatography B - Volume 843, Issue 2, 7 November 2006, Pages 240–246
نویسندگان
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