Effect of ionic strength on the pKa of ligands bound to DNA

Abstract

AbstractThe pKa of 3,8‐diamino‐6‐phenyl‐phenanthridine (DAPP), a nonquaternary analog of ethidium bromide, has been determined spectrophotometrically as a function of sodium ion concentration both free in solution and complexed to DNA. Unwinding angle determinations with this compound were determined with Col El DNA using ethidium bromide as a standard. The unwinding angle for DAPP was 24 ± 2° relative to 26° for ethidium, and this suggests that DAPP binds in a manner quite similar to ethidium and with no significant outside bound DAPP under these experimental conditions. Isobestic behavior was obtained on spectrophotometric pH titration above pH 5 as long as the ratio of DNA‐phosphate to ligand was between 100 and 300 and the DNA phosphate concentration was approximately 0.01M or greater. The loss of isosbestic behavior which occurred below pH 5 is probably due to titration of the 8 amino group of the ligand complexed to DNA. To circumvent this problem, pKa values and the extinction coefficient of the acidic species were both determined by a computer program using experimental data obtained above pH 5. The pKa of the free compound has only a minor dependence on ionic strength, while the pKa of the ligand bound to DNA in an intercalated complex depends strongly on the sodium ion concentration. The pKa of the DAPP‐DNA complex is a linear function of –log[Na+] as predicted by the ion‐condensation theory of polyelectrolytes. It was determined that DAPP is essentially completely bound to DNA under the conditions of these experiments by (1) determination of apparent pKa values as a function of total DNA concentration, (2) calculation of binding constants for the neutral species of DAPP, and (3) spectral analysis of the protonated and neutral species of DAPP bound to DNA relative to DAPP free in solution. These results support the ion‐condensation theory; provide an independent method for measuring ψ*, the average number of counterions associated per phosphate of DNA in the intercalated conformation; and illustrate that there are no specific pH effects or absolute pKa values for ligands bound to DNA, but only ionic‐strength‐dependent results.