Jahngen J H, Rossomando E F
Anal Biochem. 1983 Apr 15;130(2):406-15. doi: 10.1016/0003-2697(83)90608-5.
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.
许多参与核苷酸磷酸代谢的酶促反应（如ATP）的调节通常需要二价金属离子。在本研究中，采用反相高效液相色谱法（HPLC）研究了ATP对二价金属离子（如Mn2 +、Mg2 +和Ca2 +）的螯合作用。我们使用放射性标记的[45Ca]进行研究的结果表明，由于柱填料上的非极性基团，溶液中形成的金属 - ATP螯合物比无金属的ATP保留时间更长。两种形式ATP的回收率表明，[45Ca]仅与ATP - 金属螯合物共洗脱。其他实验表明，ATP螯合形式的保留时间不受洗脱液流速的影响，但受洗脱液pH值和甲醇浓度的影响。发现螯合形式的ATP量取决于溶液中金属的量，并且在适当条件下，即流动相中含有0.1 mM CaCl2时，也取决于二价阳离子。因此，我们发现就螯合物形成的有效性而言，金属离子的顺序为Ca2 +大于Mg2 +大于Mn2 +。螯合物的回收率及其通过HPLC的重新分析表明该络合物已经解离。通过将金属浓度恢复到其原始值可以使螯合物重新形成，并通过添加EDTA再次解离。从这些螯合物的稳定性以及反相HPLC中使用的柱填料疏水基团在增强这种稳定性中的作用方面讨论了金属螯合形式的ATP与未螯合形式的ATP的分离。
