Theoretical aspects of specific and non-specific equilibrium binding of proteins to DNA as studied by the nitrocellulose filter binding assay: Co-operative and non-co-operative binding to a one-dimensional lattice

Abstract

The analysis of equilibrium binding isotherms obtained by methods such as the nitrocellulose filter binding assay, which measure the fraction. θ, of DNA to which at least one protein molecule is bound, as a function of the free protein concentration ( L F) require a different type of theoretical framework from that required for analysis of conventional equilibrium binding data, in which the number of moles of protein bound per mole of DNA, θ c is measured as a function of L F. The theoretical framework required to analyse equilibrium binding data generated by measuring θ( L F) is developed for co-operative and non-co-operative binding of a protein to a large number of non-specific sites and to a specific sites(s) in the presence of a large number of non-specific sites on a DNA molecule. The theory is simple to apply, equations for θ( L F) being easy to derive and evaluate, and is suitable for least-squares analysis. Two examples of the application of the theory to the analysis of experimental data are provided for the specific and non-specific binding of the EcoRI restriction endonuclease to bacteriophage λ DNA, and for the specific and non-specific binding of the enzyme dihydrofolate reductase from Lactobacillus casei to pBR322 and pWDLcB1 DNA, the latter differing from the former only in a 2.9 × 10 3 base-pair insert containing the L. casei dihydrofolate reductase structural gene. The theoretical and experimental advantages and disadvantages of measuring θ( L F) rather than θ c( L F) are discussed.