The purification and characterization of deoxycytidine kinase from calf thymus

Abstract

A facile and fast approach for the purification of deoxycytidine kinase (dCK) from calf thymus was developed using a fast performance liquid chromatography system. A 73-fold enrichment of the enzyme was noted compared to unfractionated dCK. Characterization studies demonstrated that dCK had a molecular mass of 31 kDa using SDS-PAGE, an optimum pH of 7.0 and the enzyme maintained stability between 30 and 40°C. The rapid preparation of dCK demonstrated here will be valuable in the synthesis of nucleotide analogs.

Facile method for determination of deoxycytidine kinase activity in biological milieus

“…To further verify the specificity of the developed assay, the dCK inhibitor dCTP was employed, and it functions as a feedback competitor with dCK substrates such as gemcitabine for DNA polymerase [ 3 , 16 ]. As shown in Fig.…”

“…Limitations of conventional dCK assays for turbid samples such as blood or tissue debris and their low sensitivity owing to a lack of chromophores for nucleosides were resolved by the developed method. By adopting a simple purification step using anionic exchanger beads to separate dCK from biological contaminants [ 16 ], dCK activity in animal tissue can be determined easily, as shown in this study. By contrast, current methods either assay dCK in its pure state or employ filtration and chromatographic procedures to detect dCK in biological matrices.…”

“…To distinguish dCK from other kinases that may interfere with the assay, a quick purification procedure adapted by Quan et al [ 16 ] was employed. In brief, quantitated cell extracts containing dCK were incubated with 0.5 mL HiTrap Q beads (Amershan Bioscience, Uppsala, Sweden) in a 1.5 mL vial for 1 minute.…”