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  • Label-free assay based on immobilized capillary enzyme reactor of Leishmania infantum nucleoside triphosphate diphosphohydrolase (LicNTPDase-2-ICER-LC/UV).

Label-free assay based on immobilized capillary enzyme reactor of Leishmania infantum nucleoside triphosphate diphosphohydrolase (LicNTPDase-2-ICER-LC/UV).

Journal of chromatography. B, Analytical technologies in the biomedical and life sciences (2015-12-08)
Luana Magalhães, Arthur Henrique Cavalcante de Oliveira, Raphael de Souza Vasconcellos, Christiane Mariotini-Moura, Rafaela de Cássia Firmino, Juliana Lopes Rangel Fietto, Carmen Lúcia Cardoso
ABSTRACT

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 1.8mmolL(-1) for the ADP and ATP substrates, respectively. Suramin (1mmolL(-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.

MATERIALS
Product Number
Brand
Product Description

Supelco
Ascentis® Si HPLC Column, 5 μm particle size, L × I.D. 15 cm × 4.6 mm
Supelco
Ascentis® Si HPLC Column, 5 μm particle size, L × I.D. 25 cm × 4.6 mm
Supelco
Ascentis® Si HPLC Column, 5 μm particle size, L × I.D. 25 cm × 10 mm
Supelco
Ascentis® Si HPLC Column, 3 μm particle size, L × I.D. 10 cm × 3 mm
Supelco
Ascentis® Si HPLC Column, 5 μm particle size, L × I.D. 25 cm × 2.1 mm
Supelco
Ascentis® Si HPLC Column, 3 μm particle size, L × I.D. 3 cm × 2.1 mm
Supelco
Ascentis® Si HPLC Column, 3 μm particle size, L × I.D. 15 cm × 2.1 mm
Supelco
Ascentis® Si HPLC Column, 5 μm particle size, L × I.D. 15 cm × 2.1 mm
Supelco
Ascentis® Si HPLC Column, 5 μm particle size, L × I.D. 25 cm × 21.2 mm
Supelco
Ascentis® Si HPLC Column, 3 μm particle size, L × I.D. 5 cm × 2.1 mm
Supelco
Ascentis® Si HPLC Column, 5 μm particle size, L × I.D. 10 cm × 2.1 mm
Supelco
Ascentis® C8 HPLC Column, 3 μm particle size, L × I.D. 10 cm × 4.6 mm
Supelco
Ascentis® C8 HPLC Column, 5 μm particle size, L × I.D. 15 cm × 4.6 mm
Supelco
Ascentis® C8 HPLC Column, 5 μm particle size, L × I.D. 25 cm × 4.6 mm
Supelco
Ascentis® C8 HPLC Column, 3 μm particle size, L × I.D. 15 cm × 2.1 mm
Supelco
Ascentis® C8 HPLC Column, 3 μm particle size, L × I.D. 10 cm × 2.1 mm
Supelco
Ascentis® C8 HPLC Column, 3 μm particle size, L × I.D. 15 cm × 4.6 mm
Supelco
Ascentis® C8 HPLC Column, 5 μm particle size, L × I.D. 25 cm × 21.2 mm
Supelco
Ascentis® C8 HPLC Column, 3 μm particle size, L × I.D. 5 cm × 2.1 mm