Resolution of an ATP-metal chelate from metal-free ATP by reverse-phase high-performance liquid chromatography

The modulation of many enzymatic reactions involved in the metabolism of nucleotide phosphates such as ATP often require divalent metal ions. In the present study reverse-phase high-performance liquid chromatography (HPLC) was used to study the chelation of divalent metal ions, such as Mn2+, Mg2+, and Ca2+, by ATP. The results of our study using radiolabeled [45Ca] showed that the metal-ATP chelate formed in solution was retained longer than the metal-free ATP due to the nonpolar groups on the column packing. Recovery of the two forms of ATP showed that the [45Ca] coeluted exclusively with the ATP-metal chelate. Other experiments showed that the retention time of the chelated form of the ATP was unaffected by eluent flow rate, but was affected by eluant pH and methanol concentration. The amount of ATP in the chelated form was found to be dependent on the amount of the metal in solution and that under appropriate conditions, i.e., with 0.1 mM CaCl2 in the mobile phase, on the divalent cation as well. Thus, we found that in terms of effectiveness in chelate formation, the metal ions were Ca2+ greater than Mg2+ greater than Mn2+. Recovery of the chelate and its reanalysis by HPLC revealed that the complex had dissociated. The chelate could be reformed by restoring the metal concentration to its original value and dissociated again by the addition of EDTA. The resolution of the ATP in a metal chelated form from the ATP in an unchelated form is discussed in terms of the stability of these chelates and the role of the hydrophobic groups of the column packing used in the reverse-phase HPLC in enhancement of this stability.