{"title":"Fluorescent indicators for inositol 1,4,5-trisphosphate based on bioconjugates of pleckstrin homology domain and fluorescent dyes","authors":"K. Hirose, H. Takeshima, M. Iino","doi":"10.1039/A901274E","DOIUrl":null,"url":null,"abstract":"Bioconjugates of fluorescent dyes and the recombinant pleckstrin homology (PH) domain of phospholipase Cδ1 were produced with the aim of developing 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-48 to which thiol-reactive fluorescent dyes can be coupled specifically. Acrylodan- 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 (Kds) of the acrylodan- and Dapoxyl-labelled PH domains for IP3 were 659 and 586 nM, respectively. An additional mutation (C96S/V58K) in the PH domain decreased the Kds by ≡50%, providing a more sensitive method. The results indicate that these bioconjugates are promising as fluorescent indicators for IP3 quantification.","PeriodicalId":7814,"journal":{"name":"Analytical Communications","volume":"38 1","pages":"175-177"},"PeriodicalIF":0.0000,"publicationDate":"1999-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"5","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Analytical Communications","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1039/A901274E","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
引用次数: 5
Abstract
Bioconjugates of fluorescent dyes and the recombinant pleckstrin homology (PH) domain of phospholipase Cδ1 were produced with the aim of developing 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-48 to which thiol-reactive fluorescent dyes can be coupled specifically. Acrylodan- 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 (Kds) of the acrylodan- and Dapoxyl-labelled PH domains for IP3 were 659 and 586 nM, respectively. An additional mutation (C96S/V58K) in the PH domain decreased the Kds by ≡50%, providing a more sensitive method. The results indicate that these bioconjugates are promising as fluorescent indicators for IP3 quantification.