The PRECICE®dCK Phosphorylation Assay Kit, developed by NOVOCIB's R&D team, provides a ready to use solution for evaluating the phosphorylation of novel ribonucleoside and deoxyribonucleoside analogues using the active human recombinant deoxycytidine kinase supplied in the kit. Each kit includes active human recombinant deoxycytidine kinase and calibrated solution of gemcitabine to ensure accurate and reproducible phosphorylation measurements without enzymology background.
Human deoxycytidine kinase (dCK) has unusually broad specificity and is the activation enzyme for many anticancer and antiviral nucleoside analogues Human deoxycytidine kinase (dCK). It phosphorylates purine deoxynucleosides (dAR, dGR); pyrimidine deoxynucleosides (dCR); ribonucleoside (CR) and also nucleoside analogues cytarabine; gemcitabine; cladribine; fludarabine and L-nucleosides like lamivudine (3TC). Structural studies have shown how dCK accommodates both D- and L-nucleosides, explaining why it activates drugs of both chiralities.
Assay Principle
PRECICE® dCK Phosphorylation Assay Kit is based on the competitive inhibition of deoxyinosine (dIR) phosphorylation by human deoxycytidine kinase (dCK) in the presence of a nucleoside analogue.- In the absence of a nucleoside competitor, dCK phosphorylates deoxyinosine, producing dIMP.
- dIMP is immediately oxidized by IMPDH to dXMP, with concomitant formation of NADH₂.
- In the presence of a nucleoside competitor, phosphorylation of deoxyinosine (a poor dCK substrate) is inhibited, resulting in a decrease in NADH₂ formation.
Because IMPDH activity is supplied in large excess, the coupling reaction proceeds instantly, ensuring that the rate-limiting step is dCK activity. Thus, the enzymatic activity of dCK—corresponding to the formation rate of dIMP—is stoichiometrically and directly monitored by measuring NADH₂ production at 340 nm (Fig. 1).
