Label-free assay based on immobilized capillary enzyme reactor of Leishmania infantum nucleoside triphosphate diphosphohydrolase ( Lic NTPDase-2-ICER-LC/UV)
| Autor: | Raphael de Souza Vasconcellos, Juliana Lopes Rangel Fietto, Carmen Lúcia Cardoso, Rafaela de Cássia Firmino, Luana Magalhães, Christiane Mariotini-Moura, Arthur Henrique Cavalcante de Oliveira |
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| Rok vydání: | 2016 |
| Předmět: |
Immobilized enzyme
030231 tropical medicine Clinical Biochemistry NTPDase-2 01 natural sciences Biochemistry Analytical Chemistry Immobilization 03 medical and health sciences chemistry.chemical_compound Hydrolysis Bioreactors 0302 clinical medicine Animals Leishmania infantum Nucleoside-triphosphatase chemistry.chemical_classification Chromatography biology Apyrase 010401 analytical chemistry Cell Biology General Medicine Nucleoside-Triphosphatase biology.organism_classification Leishmania 0104 chemical sciences Enzyme chemistry Microscopy Electron Scanning Multidimensional enzymatic assay Nucleoside triphosphate Nucleoside triphosphate diphosphohydrolase |
| Zdroj: | LOCUS Repositório Institucional da UFV Universidade Federal de Viçosa (UFV) instacron:UFV |
| ISSN: | 1570-0232 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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