Antibody-free lanthanide-based fluorescent probe for determination of protein tyrosine kinase and phosphatase activities

A single-labeled peptide probe for measuring peptide phosphorylation status was developed by using a phosphate sensitive terbium chelate. The activity of Abl protein tyrosine kinase and T-cell protein Tyrosine phosphatase (TC PTP) was monitored in real time. To study the probe design in detail, variable substrate peptide sequences, where the enzyme target site was located from two to five amino acids apart from the nearest tyrosine residue, were synthesized. The maximum change observed in fluorescence intensity after phosphorylation was up to 320%, when the phosphorylated tyrosine was located two amino acids from the lysine coupled to the phosphate sensitive terbium chelate, demonstrating an excellent performance for a homogeneous assay. Also the longer distance of five amino acids between the phosphorylated tyrosine residue and terbium chelate resulted up to 260% change in fluorescence intensity. Figure A principle of the short peptide probe (EAI‐Y‐AAPFAK) with phosphate sensitive terbium chelate attached to the lysine side chain is described, which is proved applicable to measure in real time Abl protein tyrosine kinase and T‐cell protein tyrosine phosphatase activities. Enhancement of the terbium fluorescence could be measured upon addition of a phosphor residue to the nearby tyrosine side chain. The opposite effect could be measured, when phosphor residue is removed by protein tyrosine phosphatase.