A CE‐based assay for human protein kinase CK2 activity measurement and inhibitor screening

A new assay for protein kinase CK2 activity determination based on the quantification of a phosphorylated substrate was developed. The common CK2 substrate peptide RRRDDDSDDD, conjugated with the fluorophore 5‐[(2‐aminoethyl)amino]naphthalene‐1‐sulfonic acid at the C‐terminus served as the analyte. By means of CZE using 2 mol/L acetic acid as electrolyte and UV detection at 214 nm, the non‐phosphorylated and the phosphorylated peptide variants could be resolved within 6 min from a complex assay mixture. By this means, activity of human CK2 could be monitored by a kinetic, as well as an endpoint, method. Inhibition of human recombinant CK2 holoenzyme by 6‐methyl‐1,3,8‐trihydroxyanthraquinone and 4,5,6,7‐tetrabromobenzotriazole resulted in IC₅₀ values of 1.33 and 0.27 μM, respectively, which were similar to those obtained with the standard radiometric assay. These results suggest that the CE/UV strategy described here is a straightforward assay for CK2 inhibitor testing.