Influence of the protonation degree on the self‐association properties of adenosine 5′‐triphosphate (ATP)

The concentration dependence of the chemical shifts for the protons H‐2, H‐8 and H‐1′ of ATP has been measured in D 2 O at 27°C under several degrees of protonation in the pD range from 1.5 to 8.4. The results at pD > 4.5 are consistent with the isodesmic model of indefinite noncooperative stacking, while those at pD < 4.5 indicate a preference for the formation of dimeric stacks. The stacking tendency follows the series, ATP 4− ( K = 1.3 M −1 ) < D(ATP) 3− (2.1 M −1 ) < 1:1 ratio of D(ATP) 3− /D 2 (ATP) 2− (6.0 M −1 ) ≪ D 2 (ATP) 2− (∼ 200M −1 ) ≫ D 3 (ATP) − ( K ≃ 17 M −1 ) (for reasons of comparison all constants are expressed in the isodesmic model). These results are compared with previous data for adenosine [Ado ( K = 15 M −1 ) > 1:1 ratio of Ado/D(Ado) + (6.0 M −1 ) > D(Ado) + (0.9 M −1 )] and AMP [AMP 2− (K = 2.1 M −1 ) < D(AMP) − (3.4 M −1 ) < 1:1 ratio of D(AMP) − /D 2 (AMP) ± (5.6 M −1 ) > D 2 (AMP) ± (∼ 2M −1 ) > D 3 (AMP) + (K ≤ 1 M −1 )] to facilitate the interpretation of the results for the ATP systems. Stack formation of H 2 (ATP) 2− is clearly favored by additional ionic interactions; this is confirmed by measuring via potentiometric pH titrations the acidity constants of H 2 (ATP) 2− in solutions containing different concentrations of ATP. It is suggested that in the [H 2 (ATP)] 4− 2 dimer intermolecular ion pairs (and hydrogen bonds) are formed between the H + (N‐1) site of one H 2 (ATP) 2− and the γ‐P(OH)(O) − 2 group of the other; in this way (a) the stack is further stabilized, and (b) the positive charges at the adenine residues are compensated (otherwise repulsion would occur as is evident from the adenosine systems). A detailed structure for the [H 2 (ATP)] 4− 2 ‐dimer is proposed and some implications of the described stacking properties of ATP for biological systems are indicated.