کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
1199123 1493519 2015 6 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
A capillary electrophoresis method with dynamic pH junction stacking for the monitoring of cerebroside sulfotransferase
موضوعات مرتبط
مهندسی و علوم پایه شیمی شیمی آنالیزی یا شیمی تجزیه
پیش نمایش صفحه اول مقاله
A capillary electrophoresis method with dynamic pH junction stacking for the monitoring of cerebroside sulfotransferase
چکیده انگلیسی


• The first validated CE method for monitoring CST activity was developed.
• A LOD for PAP of 66.6 nM was achieved by dynamic pH junction stacking.
• The new assay is suitable for enzyme characterization and inhibitor screening.
• The method will help to develop drugs for MLD, a rare and severe genetic disease.

Metachromatic leukodystrophy (MLD) is a rare and severe genetic disease. Inhibition of cerebroside sulfotransferase (CST) has been proposed as a promising new therapeutic strategy for the treatment of MLD. CST catalyzes the transfer of a sulfate group from the coenzyme 3′-phosphoadenosine-5′-phosphosulfate (PAPS) to cerebroside yielding cerebroside sulfate and adenosine-3′,5′-diphosphate (PAP). So far only a few weak CST inhibitors have been described. The goal of the present study was to establish a suitable assay for identifying and characterizing novel CST inhibitors. To this end, we developed and optimized a capillary electrophoresis (CE) based assay for monitoring the catalytic activity of CST by measuring the formation of PAP. A sample matrix consisting of 5 mM phosphate buffer with about 0.0001% polybrene at pH 7.4 and a background electrolyte (BGE) containing 75 mM phosphate buffer with 0.002% polybrene at pH 5.6 were utilized to achieve a stacking effect for PAP by dynamic pH junction. This led to a limit of detection for the enzymatic product PAP of 66.6 nM. The CE method was sensitive, robust, and suitable for CST inhibitor screening, Ki value determination, and enzyme kinetic studies. Selected reference compounds were tested in order to validate the assay, including the substrates cerebroside and psychosine, and the inhibitor Congo Red. The newly developed CE method will be useful for the identification and development of novel CST inhibitors which are urgently needed for the treatment of MLD.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Chromatography A - Volume 1407, 14 August 2015, Pages 222–227
نویسندگان
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