A Direct OnFlow Assay to Monitor the Activity of Purine Nucleoside Phosphorylase from Mycobacterium tuberculosis

Abstract

Purine nucleoside phosphorylase (PNP) catalyzes the phosphorolysis of N-ribosidic bonds of purine nucleosides and deoxynucleosides to the corresponding purine bases and (deoxy)ribosyl-1-phosphate. PNP plays a central role in purine recycling and salvage pathway and has been considered an attractive chemotherapeutic target for several diseases. In this work, PNP from Mycobacterium tuberculosis (MtPNP) was covalently immobilized into fused-silica capillaries. The activity of the produced immobilized enzyme reactor (MtPNP-IMER) was monitored onflow in a multidimensional liquid chromatography system containing the MtPNP-IMER in the first dimension. A C18 analytical column inserted in the second dimension furnished the rapid chromatographic separation of the substrate (inosine) and product (hypoxanthine) from the MtPNP catalyzed reaction, allowing monitoring MtPNP-IMER activity through the direct quantification of hypoxanthine formed. Kinetic studies showed that the K_M values (59.19 μmol L⁻¹) for substrates using the immobilized enzyme were consistent with those reported for the free enzyme in solution (40 μmol L⁻¹). To validate the use of the proposed methodology for screening purposes, a fourth-generation immucillin derivative (DI4G), known as a PNP inhibitor, was used as a standard inhibitor. The studies revealed that the immobilized enzyme retained the ability to recognize enzyme inhibitors and the onflow assay allowed the characterization of the inhibitor by determining the IC₅₀ (29.85 ± 1.02 nmol L⁻¹), inhibition mechanism and constant (K_i = 34.8 ± 0.2 nmol L⁻¹). The results revealed that the immobilized MtPNP retained its catalytic activity (up to 60%) and the ability to recognize ligands with high stability for up to 10 days. The proposed model has as advantages the automation and the possibility of reusing the same amount of enzyme in several assays.