A confocal Raman microspectrometer was used to investigate the influence of Ca2+ cations on low pH-induced DNA structural changes. The effects of Ca2+ cations on the protonation mechanism of opening AT and changing the protonation of GC base pairs in DNA are discussed. Based on the observation that the midpoint of the transition of Watson-Crick GC base pairs to protonated GC base pairs lies at around pH 3 (analyzing the 681 cm-1 line), measurements were carried out on calf thymus DNA at neutral pH and pH 3 in the presence of low and high concentrations of Ca2+ cations. Raman spectra show that low concentrations of Ca2+ cations partially protect DNA against protonation of cytosine (characteristic line at 1262 cm-1) and do not protect adenine (characteristic line at 1304 cm-1) and the N(7) of guanine (line at 1488 cm-1) against binding of H+. High Ca2+ concentrations can prevent protonation of cytosine and protonation of adenine (little disruption of AT pairs). Analyzing the line at 1488 cm-1, which obtains most of its intensity from a guanine vibration, high salt was also found to protect the N(7) of guanine against protonation.
- Raman microspectroscopy