A Robust Method for Determining DNA Binding Constants Using Capillary Zone Electrophoresis

Abstract

Capillary zone electrophoresis (CZE or CE) with on-line UV detection was utilized to measure the binding constants between purified calf thymus DNA and a library of designed tetrapeptides which had been constructed using unnatural amino acids with thiazole ring side chains. Mixtures containing a constant amount of a tetrapeptide, the neutral marker (mesityl oxide), and varying concentrations of DNA were prepared and equilibrated at 8°C for 12 h. CE was then utilized to separate unbound tetrapeptides from the DNA–peptide complex. The UV absorbance of the peak representing unbound tetrapeptide decreased incrementally as a result of increasing the concentration of DNA in the equilibrium mixture. The absorbance of the peak corresponding to the unbound tetrapeptide was obtained directly from the electropherogram and used in the calculation of the DNA–peptide binding constants. The binding constant for each tetrapeptide to calf thymus DNA was obtained from the negative slope of a Scatchard plot and a comparison of the binding constants for different peptides showed that the tetrapeptides in the library have DNA-binding affinities ranging from 102to 106M−1.