کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
1214826 1494043 2016 10 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Label-free assay based on immobilized capillary enzyme reactor of Leishmania infantum nucleoside triphosphate diphosphohydrolase (LicNTPDase-2-ICER-LC/UV)
موضوعات مرتبط
مهندسی و علوم پایه شیمی شیمی آنالیزی یا شیمی تجزیه
پیش نمایش صفحه اول مقاله
Label-free assay based on immobilized capillary enzyme reactor of Leishmania infantum nucleoside triphosphate diphosphohydrolase (LicNTPDase-2-ICER-LC/UV)
چکیده انگلیسی


• Nucleoside triphosphate diphosphohydrolase (NTPDase-2) from Leishmania infantum immobilized onto a fused silica capillary.
• Label free assay by using LiNTPDase-2-ICER-LC/UV system.
• Applicability of the method developed herein for the identification of new ligands.

Nucleoside triphosphate diphosphohydrolase (NTPDase) is an enzyme belonging to the apyrase family that participates in the hydrolysis of the nucleosides di- and triphosphate to the corresponding nucleoside monophosphate. This enzyme underlies the virulence of parasites such as Leishmania. Recently, an NTPDase from Leishmania infantum (LicNTPDase-2) was cloned and expressed and has been considered as a new drug target for the treatment of leishmaniasis. With the intent of developing label-free online screening methodologies, LicNTPDase-2 was covalently immobilized onto a fused silica capillary tube in the present study to create an immobilized capillary enzyme reactor (ICER) based on LicNTPDase-2 (LicNTPDase-2-ICER). To perform the activity assays, a multidimensional chromatographic method was developed employing the LicNTPDase-2-ICER in the first dimension, and an analytical Ascentis C8 column was used in the second dimension to provide analytical separation of the substrates and products. The validated LicNTPDase-2-ICER method provided the following kinetic parameters of the immobilized enzyme: KM of 2.2 and 1.8 mmol L−1 for the ADP and ATP substrates, respectively. Suramin (1 mmol L−1) was also shown to inhibit 32.9% of the enzymatic activity. The developed method is applicable to kinetic studies and enables the recognition of the ligands. Furthermore, a comparison of the values of LicNTPDase-2-ICER with those obtained with an LC method using free enzyme in solution showed that LicNTPDase-2-ICER-LC/UV was an accurate and reproducible method that enabled automated measurements for the rapid screening of ligands.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Chromatography B - Volume 1008, 1 January 2016, Pages 98–107
نویسندگان
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