Lennon A J, Scott N R, Chapman B E, Kuchel P W
Department of Biochemistry, University of Sydney, New South Wales, Australia.
Biophys J. 1994 Nov;67(5):2096-109. doi: 10.1016/S0006-3495(94)80693-9.
The diffusion coefficient (D) of 2,3-bisphosphoglycerate (DPG) was measured using pulsed-field gradient (PFG)-31P nuclear magnetic resonance spectroscopy in solutions containing 2.7-5.0 mM hemoglobin (Hb) and a range of DPG concentrations. The dependence of the measured values of D on the fraction of the total DPG in the sample that is bound to Hb enabled the estimation of the dissociation constants (Kd) of complexes of DPG with carbonmonoxygenated, oxygenated, and deoxygenated Hb; the values of Kd (mM), measured at 25 degrees C, pH 6.9 and in 100 mM bis Tris/50 mM KCl, were 1.98 +/- 0.26, 1.8 +/- 0.5 and 0.39 +/- 0.26, respectively. In intact erythrocytes the apparent diffusion coefficient, Dapp, of DPG was larger in oxygenated and carbonmonoxygenated cells (6.17 +/- 0.20 x 10(-11) m2s-1) than in deoxygenated cells (4.10 +/- 0.23 x 10(-11) m2s-1). Changes in intracellular DPG concentration (5-55 mM) in erythrocytes, brought about by incubation in a medium containing inosine and pyruvate, did not result in significant changes in the value of Dapp; this result supports the hypothesis that DPG binds to other sites in the erythrocyte. Monte Carlo simulations of diffusion in biconcave discs were used to test the adequacy of the values of Kd estimated in solution to describe the binding of DPG to Hb in oxygenated and deoxygenated erythrocytes. The results of the simulations implied that the value of Kd estimated for deoxygenated Hb-DPG was greater than expected from the experiments involving intact erythrocytes. This difference is surmised to be at least partly due to the difficulty of measuring D at low-ligand concentrations. Notwithstanding this shortcoming, the PFG method appears to be suitable for probing interactions between macromolecules and ligands when the Kd is in the millimolar range. It is one of the few techniques available in which these interactions can be studied in intact cells. In addition, the Monte Carlo simulations of the diffusion experiments highlighted important differences between theory and experiment relating to the nature of molecular motion inside the cells.
使用脉冲场梯度（PFG）-31P核磁共振波谱法，在含有2.7 - 5.0 mM血红蛋白（Hb）和一系列2,3-二磷酸甘油酸（DPG）浓度的溶液中测量了DPG的扩散系数（D）。所测D值对样品中与Hb结合的总DPG分数的依赖性，使得能够估算DPG与一氧化碳化血红蛋白、氧合血红蛋白和脱氧血红蛋白复合物的解离常数（Kd）；在25℃、pH 6.9以及100 mM双三羟甲基氨基甲烷/50 mM氯化钾条件下测得的Kd值（mM）分别为1.98±0.26、1.8±0.5和0.39±0.26。在完整红细胞中，氧合和一氧化碳化细胞中DPG的表观扩散系数Dapp（6.17±0.20×10-11 m2s-1）大于脱氧细胞中的（4.10±0.23×10-11 m2s-1）。通过在含有肌苷和丙酮酸的培养基中孵育，使红细胞内DPG浓度（5 - 55 mM）发生变化，并未导致Dapp值出现显著变化；这一结果支持了DPG与红细胞中其他位点结合的假说。采用双凹圆盘扩散蒙特卡罗模拟来检验在溶液中估算的Kd值，是否足以描述DPG在氧合和脱氧红细胞中与Hb的结合情况。模拟结果表明，估算的脱氧Hb-DPG的Kd值高于涉及完整红细胞的实验预期值。推测这种差异至少部分是由于在低配体浓度下测量D存在困难。尽管存在这一缺点，但当Kd处于毫摩尔范围时，PFG方法似乎适用于探究大分子与配体之间的相互作用。它是少数几种能够在完整细胞中研究这些相互作用的可用技术之一。此外，扩散实验的蒙特卡罗模拟突出了理论与实验在细胞内分子运动性质方面的重要差异。
