Single-Label Kinase and Phosphatase Assays for Tyrosine Phosphorylation Using Nanosecond Time-Resolved Fluorescence Detection

Abstract

The collision-induced fluorescence quenching of a 2,3-diazabicyclo[2.2.2]oct-2-ene-labeled asparagine (Dbo) by hydrogen atom abstraction from the tyrosine residue in peptide substrates was introduced as a single-labeling strategy to assay the activity of tyrosine kinases and phosphatases. The assays were tested for 12 different combinations of Dbo-labeled substrates and with the enzymes p60c-Src Src kinase, EGFR kinase, YOP protein tyrosine phosphatase, as well as acid and alkaline phosphatases, thereby demonstrating a broad application potential. The steady-state fluorescence changed by a factor of up to 7 in the course of the enzymatic reaction, which allowed for a sufficient sensitivity of continuous monitoring in steady-state experiments. The fluorescence lifetimes (and intensities) were found to be rather constant for the phosphotyrosine peptides (ca. 300 ns in aerated water), while those of the unphosphorylated peptides were as short as 40 ns (at pH 7) and 7 ns (at pH 13) as a result of intramolecular quenching. Owing to the exceptionally long fluorescence lifetime of Dbo, the assays were alternatively performed by using nanosecond time-resolved fluorescence (Nano-TRF) detection, which leads to an improved discrimination of background fluorescence and an increased sensitivity. The potential for inhibitor screening was demonstrated through the inhibition of acid and alkaline phosphatases by molybdate.

Fluorescence in Nanobiotechnology: Sophisticated Fluorophores for Novel Applications

“…Using a fluorazaphore‐labeled fluorescent amino acid (the asparagine derivative Fmoc‐DBO), intramolecular contact formation rates in biopolymers could be determined 59. Further promising applications are for instance the investigation of fluorescence quenching in kinase and phosphatase assays by time‐resolved techniques 60. Although in principle both steady‐state and time‐resolved techniques can be employed, long lifetime fluorophores offer the advantage of a high degree of background fluorescence suppression, when time‐gating is used.…”

Study on the binding of 2,3-diazabicyclo[2.2.2]oct-2-ene with bovine serum albumin by fluorescence spectroscopy

“…Therefore, fluorescence quenching can be considered as a sensitive technique to monitor protein fluorescent probe interactions. Critical literature survey reveals that attempts have not been made so far to investigate the mechanism of interaction of azoalkane [2,3- The DBO fluorophore has already been extensively used as a fluorescent probe to study its inter and intramolecular fluorescence quenching by biomolecular compounds such as tryptophan and tyrosine [9][10][11], the nucleobase guanine [12,13], and antioxidants [14][15][16]. More recently, DBO has also been employed as a fluorescence energy acceptor [17][18][19][20], e.g., to investigate fluorescence energy transfer from the amino acid Trp as donor in peptides [17][18][19][20].…”

Selective Sensing of Tyrosine Phosphorylation in Peptides Using Terbium(III) Complexes

“…For many years, a number of laboratories developed elegant optical sensors to evaluate the activities of these enzymes. In some of them, substrate peptide was conjugated (or fused) to a probe molecule (e.g., Tb(III) complexes [ 35 – 40 ], Mg(II) complexes [ 41 – 47 ], Ca(II) complex [ 48 ], Zn(II) complex [ 49 ], Cd(II) complex [ 50 ], peptide derivatives [ 51 , 52 ], and others [ 53 , 54 ]). The other sensors involve noncovalent interactions between a substrate and a probe (e.g., Tb(III) ion [ 55 – 62 ], Eu(III) complex [ 63 , 64 ], platinum(II) complex [ 65 ], and Tb(III) complexes [ 66 – 69 ]).…”