Protonation of cytosine in DNA

Spectrophotometric acid titrations of DNA samples of different GC content were performed at different wavelengths. From the acid titration profile and absorbance changes of deoxycytidine-5′-monophosphate and DNA the extent of protonated cytosine within the DNA double-stranded molecule was estimated (pK 3.65 at 25°C. in 0.02M KCl). At constant counterion concentration and temperature the maximum of protonated cytosine in DNA before denaturation occurs depends on the base composition and can exceed 50%. The thermal stability of the DNA secondary structure is strongly reduced with increasing amount of ionized cytosine residues. The degree of protonation of cytosine in DNA is decreased with increasing counterion concentration.