Thermodynamic bookkeeping when nucleotides bind. Applications of the theory of linked functions

The thermodynamic theory of linked functions was used to determine the numbers of modifier ions involved when nucleotides dissociate. Nucleotide dissociation constants, obtained spectrophotometrically using Dowex-1 resin as a model system, were plotted on log/log paper with respect to the modifier concentrations. The slopes of the lines represent the net number of modifier molecules/ions involved in the dissociation. Varying numbers of nucleotides are bound to the resin because the resin capacity is determined by the total number of charges bound. The nucleotides bind to the resin at comparable diffusion-limited rates, irrespective of how tightly they bind. When ATP binds at pH 6.8, 4 chlorides, 4 formates, 2 succinates or 1.4 citrates are displaced, indicating that the fully charged (ATP4-) nucleotide binds. By comparing ATP, ADP and AMP it was possible to evaluate the contributions of the adenosine moiety and each phosphate to the binding. Between pH 2 and 3, where ATP has two negative charges, ATP binds largely as the trianion, displacing 2.7 chlorides and 0.7 protons. In the presence of 4 mM magnesium, 0.58 magnesiums facilitate the dissociation by chelating 58% of the liberated ATP. Calcium behaved similarly to magnesium but aluminum, at pH 6.8, promoted the binding of ATP as an (A1.ATP)3- complex with the concomitant liberation of three chloride ions. These experimental thermodynamic stoichiometries were found to be independent of the concentrations of the other modifiers present. Thermodynamic linkage stoichiometries can be evaluated from log K vs. log (modifier) plots when a direct determination of modifier binding is impossible.(ABSTRACT TRUNCATED AT 250 WORDS)