A phosphotyrosine-containing quenched fluorogenic peptide as a novel substrate for protein tyrosine phosphatases

Mca-Gly-Asp-Ala-Glu-Tyr(PO3H2)-Ala-Ala-Lys(DNP)-Arg-NH2, where Mca is (7-me thoxycoumarin-4-yl)acetyl and DNP is 2,4-dinitrophenyl, was synthesized as a fluorogenic substrate for protein tyrosine phosphatases (PTPs), In the pe ptide, the fluorescent Mca group is quenched efficiently by the DNP group. Although,the fluorescence intensity of the substrate was practically;unchan ged upon PTP-catalysed dephosphorylation, it increased approx. 120-fold upo n subsequent treatment with chymotrypsin. Analysis by HPLC showed that chym otrypsin cleaved only the dephosphorylated substrate at the Tyr-Ala bond. T hus with the aid of chymotrypsin, dephosphorylation of the substrate can be measured fluorometrically. A strictly linear correlation was observed betw een PTP concentration and dephosphorylation rate. The fluorogenic substrate was dephosphorylated by some PTPs much more rapidly than the corresponding P-32-labelled substrate used for comparison, whereas alkaline phosphatase dephosphorylated the two substrates at similar rates. The fluorogenic subst rate is therefore more specific for PTPs than the radiolabelled substrate. The assay with the fluorogenic substrate could be applied to the estimation of kinetc parameters and measurement of PTP activity in crude-enzyme prepa rations. The lower detection limit of our assay (1 mu M substrate in 200 mu l of reaction mixture) was estimated to be 0.2-0.4 pmol, whereas it was es timated to be about 1 pmol in the assay that used P-32-labelled peptide (sp ecific radioactivity of approx. 1000 c.p.m./pmol). Our assay is simple, spe cific, highly sensitive and non-radioisotopic, and hence would contribute g reatly to the development of PTP biology.