Quantitative analysis of cation binding to the adenosine nucleotides using the Variable Ionic Strength method: Validation of the Debye–Hückel–Onsager theory of electrophoresis in the absence of counterion binding

Abstract

The free solution mobilities of the adenosine nucleotides 5'-adenosine triphosphate (ATP), 5'-adenosine diphosphate (ADP), 5'-adenosine monophosphate (AMP), and 3'-5'-cyclic AMP (cAMP) have been measured in diethylmalonate buffers containing a wide variety of monovalent cations. The mobilities of all nucleotides increase gradually with the increase in intrinsic conductivity of the cation in the BGE. However, at a given conductivity, the mobilities observed for ATP, ADP, and AMP in BGEs containing alkali metal ions and other cations are lower than these observed in BGEs containing tetraalkylammonium ions. Since the mobility of cAMP is independent of the cation in the BGE, the results suggest that the relatively low mobilities observed for ATP, ADP, and AMP in BGEs containing cations other than a tetraalkylammonium ion are due to cation binding, reducing the effective net charge of the nucleotide and thereby reducing the observed mobility. To measure the binding quantitatively, the mobilities of the nucleotides were measured as a function of ionic strength. The mobilities of ATP, ADP, and AMP decrease nonlinearly with the square root of ionic strength (I(1/2)) in BGEs containing an alkali metal ion or Tris(+). By contrast, the mobilities decrease linearly with I(1/2) in BGEs containing a nonbinding quaternary ammonium ion, as expected from Debye-Hückel-Onsager (DHO) theory. The mobility of cAMP, a nonbinding analyte, decreases linearly with I(1/2), regardless of the cation in the BGE. Hence, a nonlinear decrease of the mobility of an analyte with I(1/2) appears to be a hallmark of counterion binding. The curved mobility profiles observed for ATP, ADP, and AMP in BGEs containing an alkali metal ion or Tris(+) were analyzed by nonlinear curve fitting, using difference mobility profiles to correct for the effect of the physical properties of BGE on the observed mobilities. The calculated apparent dissociation constants range from 22 to 344 mM, depending on the particular cation-nucleotide pair. Similar values have been obtained by other investigators, using different methods. Interestingly, Tris(+) and Li(+) bind to the adenosine nucleotides with approximately equal affinities, suggesting that positively charged Tris(+) buffer ions can compete with alkali metal ions in Tris-buffered solutions.

Quantitative Analysis of Monovalent Counterion Binding to Random-Sequence, Double-Stranded DNA Using the Replacement Ion Method

“…The mobilities observed for ATP, ADP and AMP in solutions containing tetraalkylammonium ions are higher than observed in solutions containing other cations with the same intrinsic conductivity, while the mobilities of cAMP and A-COOH are independent of the nature of the cation (44). Hence, ATP, ADP and AMP bind all (tested) monovalent cations except the quaternary ammonium ions, in agreement with other results in the literature (42,43,45).…”

“…Very similar results have been observed with the adenosine nucleotides (44). The mobilities observed for ATP, ADP and AMP in solutions containing tetraalkylammonium ions are higher than observed in solutions containing other cations with the same intrinsic conductivity, while the mobilities of cAMP and A-COOH are independent of the nature of the cation (44).…”

“…The mobility profiles observed with TBA + as the non-binding ion are illustrated in Figure 2; similar results were obtained with TPA + (data not shown). The mobility of cAMP increases linearly with increasing [Li + ] due to the conductivity effect, as expected since cAMP does not bind monovalent cations (44) and the intrinsic conductivity of Li + is greater than that of TBA + (58). By contrast, the mobilities of ATP and ADP decrease significantly with increasing [Li + ] at the beginning of the titration, due to counterion binding.…”

“…The binding affinities increase with the increasing number of phosphate residues in the nucleotide and decrease with the increasing radius of the bound cation, as expected for an electrostatic interaction between analyte and ligand (43,45). Tris + buffer ions also bind to the adenosine nucleotides, with apparent dissociation constants similar to those observed for Li + (44). Since Tris + and Li + differ markedly in size, the binding of Tris + probably occurs through a combination of hydrogen bonds and electrostatic interactions (44,45).…”

Do zwitterions contribute to the ionic strength of a solution?

“…However, capillary electrophoresis measurements in free solution are ideal for this purpose. If an analyte, such as a small DNA oligomer, does not bind one of the ions in the background electrolyte, its electrophoretic mobility will decrease as the square root of ionic strength [ 9 ], as predicted by Debye-Hückel-Onsager theory of electrophoresis [ 10 ], regardless of whether the ionic strength is increased by increasing the buffer concentration or by adding a neutral salt to the buffer. The same result would be expected upon adding a zwitterion to the buffer, if zwitterions contribute to the ionic strength.…”