Fluorescent indicators for inositol 1,4,5-trisphosphate based on bioconjugates of pleckstrin homology domain and fluorescent dyes

Bioconjugates of fluorescent dyes and the recombinant pleckstrin homology ( PH) domain of phospholipase C delta(1) were produced with the aim of develo ping a method to quantify inositol 1,4,5-trisphosphate (IP3) in biological samples. We replaced Cys-96 of the PH domain with Ser while retaining Cys-4 8 to which thiol-reactive fluorescent dyes can be coupled specifically. Acr ylodan- and Dapoxyl-labelled C96S PH domain mutants exhibited fluorescence upon UV illumination with an emission peak at wavelengths of 505 and 514 nm , respectively. IP3 induced decreases in the fluorescence intensity with a red shift in the emission spectra. The dissociation constants (K(d)s) Of th e acrylodan- and Dapoxyl-labelled PH domains for IP3 were 659 and 586 nM, r espectively. An additional mutation (C96S/V58K) in the PH domain decreased the K(d)s by similar to 50%, providing a more sensitive method. The results indicate that these bioconjugates are promising as fluorescent indicators for IP3 quantification.