NONISOTOPIC QUANTITATIVE-ANALYSIS OF PROTEIN-DNA INTERACTIONS AT EQUILIBRIUM

Two versions of an enzyme-linked immunosorbent assay-type method to qu antify protein-DNA interactions at equilibrium were developed. The fir st variant comprised immobilization of DNA-binding protein on microtit er plates, incubation with biotinylated DNA, and tagging of bound DNA with streptavidin- and biotin-substituted horseradish peroxidase. In t he second version, biotinylated DNA was immobilized on streptavidin-su bstituted microtiter plates, incubated with DNA-binding protein, and b ound protein was quantified with specific antibodies. To illustrate th e method, the interaction of a fusion protein between glutathione-S-tr ansferase and the DNA-binding domain of the helicase-like transcriptio n factor with its cis-element (the B box of the plasminogen activator inhibitor-1 promoter) was determined with both versions: a 1:1 stoichi ometric interaction with an equilibrium dissociation constant (K-d) Of 1 nM was found, which is similar to the value determined by electroph oretic mobility shift assay, demonstrating the validity of the assays. (C) 1997 Academic Press.