Schliselfeld L H, Burt C T, Labotka R J
Biochemistry. 1982 Jan 19;21(2):317-20. doi: 10.1021/bi00531a018.
The 31P NMR proton-decoupled spectra of alpha, beta-methylene-ATP [Ap(CH2)pp], beta, gamma-methylene-ATP [App(CH2)p], and alpha, beta-methylene-ADP [Ap(CH2)p] were measured as functions of pH and Mg2+ concentration. Each ATP analogue yielded three resonances: two doublets and one doublet-of-a-doublet. Assignments of resonances were based upon spin-coupling multiplets, their coupling constant magnitudes (24-27 Hz for -P-O-P- and 4-10 Hz for -P-CH2-P-), and the magnitude of the chemical shift movement during proton titration or its direction of movement. All phosphonyl resonances are substantially downfield compared to phosphoryl resonances. The chemical shifts of terminal phosphonyl units moved upfield with increasing pH or rising Mg2+ concentration. The chemical shifts of phosphonyl and phosphoryl anhydride plus ester units usually either moved downfield during proton titration and addition of Mg2+ or remained constant. Accurate pKa' values were readily determined from chemical shift movements as a function of pH: 3.05 +/- 0.04 and 8.80 +/- 0.05 for App(CH2)p, 7.34 +/- 0.06 for Ap(CH2)pp, and 8.29 +/- 0.02 for Ap(CH2)p. Addition of Mg2+ or Tris produced an acidic shift of the alkaline pKa' values. Addition of Mg2+ at pH 7.0 to the nucleotides caused large movements in the chemical shifts of their terminal two phosphorus atoms.
测定了α,β-亚甲基-ATP [Ap(CH2)pp]、β,γ-亚甲基-ATP [App(CH2)p]和α,β-亚甲基-ADP [Ap(CH2)p]的31P NMR质子去耦谱随pH值和Mg2+浓度的变化。每种ATP类似物产生三个共振峰：两个双峰和一个双二重峰。共振峰的归属基于自旋耦合多重峰、它们的耦合常数大小（-P-O-P-为24 - 27 Hz，-P-CH2-P-为4 - 10 Hz）以及质子滴定过程中化学位移移动的大小或其移动方向。与磷酰基共振相比，所有膦酰基共振都明显位于低场。末端膦酰基单元的化学位移随pH值升高或Mg2+浓度增加而向高场移动。膦酰基和磷酰酸酐加酯单元的化学位移通常在质子滴定和添加Mg2+过程中向低场移动或保持不变。根据化学位移随pH值的变化很容易确定准确的pKa'值：App(CH2)p为3.05±0.04和8.80±0.05，Ap(CH2)pp为7.34±0.06，Ap(CH2)p为8.29±0.02。添加Mg2+或Tris会使碱性pKa'值发生酸性位移。在pH 7.0时向核苷酸中添加Mg2+会导致其末端两个磷原子的化学位移发生大幅移动。
