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An equation to model fluorescent probe displacement by non‐fluorescent competitive ligands
Author(s) -
Beckmann Joe D.,
Martin Emily D.,
Gaffney Kristen
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.962.6
Subject(s) - titration , chemistry , fluorescence , ligand (biochemistry) , nucleotide , competitive binding , stereochemistry , receptor , biochemistry , physics , quantum mechanics , gene
Measurement of ligand binding to a receptor to evaluate K d values is fundamental. If binding is spectrally silent, then a probe with dynamic optical properties can be employed. Such is the case in our studies of nucleotides binding to phenol sulfotransferase, SULT1A1, one such enzyme that requires 3’‐phosphoadenosine‐ 5’‐phosphosulfate (PAPS). Whereas binding of PAP(S) and other nucleotides is spectrally invisible, 3’‐trinitrophenylated‐adenylates (e.g. TNP‐AMP) form a fluorescent SULT:TNP‐AXP complex. These TNP probes have seen considerable applications for kinases and other ATP‐binding proteins. However, many published analyses of probe displacement by biological nucleotides are confounded by assumptions and questionable analyses. We have derived an equation that predicts binding constants by adequately modeling competition fluorimetric titrations, and have applied it to both published works and to our SULT analyses. The procedure requires titration with the probe and determination of its K d value, followed by competitive titration with another ligand. Parameters required or derived are: total receptor, probe, and ligand concentrations; K d (probe), K d (ligand), F max , and observed F values. We see agreement between K d values obtained by this method and by kinetic and isothermal titration methods, for the binding of PAP to recombinant bovine SULT1A1.