A multidimensional liquid chromatography method for monitoring on flow the activity of PNP-Mt immobilized enzyme

Purine Nucleoside Phosphorylase (PNP) is a key enzyme in the purine salvage pathway and catalyzes the cleavage of deoxyribonucleosides to the corresponding purine base and sugar-1-phosphate in presence of inorganic phosphate (Pi). PNP from Mycobacterium tuberculosis (MtPNP) has been considered an attractive target for drug development against tuberculosis [1]. Therefore, the development of analytical tools for the fast identification of specific inhibitors for this enzyme has become an interesting task. A promising approach is the use of immobilized enzymes reactors (IMERs) for on-flow screening studies. MtPNP-IMER was prepared by the covalent immobilization onto a fused silica capillary, based on previously published conditions for human PNP [2]. For the on-flow multidimensional chromatography activity assay, the MtPNP-IMER was inserted in the first dimension (HEPES 100 mM pH 7.4, 0.05 mL/min) and an octyl column (Phenomenex Chrospher RP-8, 150 x 4.6 mm, 5 uM, 100 Å) in the second dimension. Inosine and the formed hypoxanthine, respectively substrate and product of the enzymatic reaction, were separated in the analytical column by the use of a mobile phase containing 1% TEA pH 6.0:MeOH (95:5), at a flow rate 0.8 mL/min, and λ = 280nm. The estimated amount of MtPNP immobilized onto each capillary was 130.5 ± 32.3 μg of total protein, indicating that the immobilization procedure was efficient. The MtPNP activity was monitored by the direct quantification of formed hypoxanthine. Kinetic studies showed that the KM value for the substrate inosine using the immobilized enzyme (41μM) is similar to that one measured for the enzyme in solution (40±3μM). These results revealed that the immobilized MtPNP retained its catalytic activity and represents a useful way of reutilizing the same amount of enzyme in diverse assays, including in the screening of new MtPNP inhibitors