Proton nmr spectroscopy is used to measure the deuterium exchange rates of C8 protons in individual purines of the dodecamer 5'-d( CGCGAATTCGCG) -3' and their temperature dependence. In perfect agreement with results from tritium labeling and laser Raman spectroscopy, we find that the DNA secondary structure retards the rates of purine C8H exchange. The largest effects are observed for the C8 protons of adenines whose rates of exchange at 40°C are 3-to 4-fold lower than that in 5'-adenosine monophosphate. Moreover, the retardation of exchange at the central adenine is greater than that at its 5'-neighbor. For the guanines, the exchange rates are up to 2-fold lower than that in 5'-guanosine monophosphate, and the largest retardation is observed for the bases a t positions 10 and 12. A dependence on base sequence is also observed for the activation energy for exchange.
The activation energy is largest for the adenines and its value is 4 kcal/mol higher than that in 5'-adenosine monophosphate. The lowest activation energy is observed for the guanine in position 4 and the value is the same as in 5'-guanosine monophosphate. These results demonstrate the sensitivity of the purine C8H exchange kinetics to sequence-dependent conformational features of B-DNA in solution state.