Homogeneous assay for enzymatic activity

Abstract

An assay is disclosed for measuring activity of enzymes, such as kinases, phosphatases, and proteses. Measurements of enzymatic activity are accomplished in a homogenous assay format utilizing a fluorescence quenching technique employing paramagnetic metal ions.

1 . A composition formed in a fluorescence quench-based homogenous assay for enzymatic activity comprising
 a paramagnetic metal ion and   a substrate for an enzyme or an enzymatic endproduct resulting from reaction of the enzyme with the substrate, the substrate or endproduct containing
 a fluorophore label and 
 containing a target group to which the paramagnetic metal ion is bound to form a complex of the target and ion, 
   said complex being in proximity to the fluorophore to cause the specific quenching of the fluorescence of the label when the complex forms.   
     
     
         2 . The composition of  claim 1  wherein the target group is a phosphoryl group. 
     
     
         3 . The composition of  claim 1  wherein the target group is an imidazole group. 
     
     
         4 . The composition of  claim 1  wherein the paramagnetic metal ion is Fe (III). 
     
     
         5 . The composition of  claim 1  wherein the paramagnetic metal ion is Ni (II). 
     
     
         6 . The composition of  claim 2  wherein the paramagnetic metal ion is Fe (III). 
     
     
         7 . The composition of  claim 3  wherein the paramagnetic metal ion is Ni (II). 
     
     
         8 . The composition of  claim 1  wherein the substrate or endproduct contains a single fluorophore label which is the only dye entity attached thereto. 
     
     
         9 . The composition of  claim 6  wherein the substrate or endproduct contains a single fluorophore label which is the only dye entity attached thereto 
     
     
         10 . The composition of  claim 7  wherein the substrate or endproduct contains a single fluorophore label which is the only dye entity attached thereto. 
     
     
         11 . The composition of  claim 1  wherein the paramagnetic metal ion, in addition to being bound to be the target group, is coordinated with a chelator. 
     
     
         12 . The composition of  claim 6  wherein the paramagnetic metal ion, in addition to being bound to be the target group, is coordinated with a chelator. 
     
     
         13 . The composition of  claim 7  wherein the paramagnetic metal ion, in addition to being bound to be the target group, is coordinated with a chelator.