Martino A J, Ferrone F A
Department of Physics and Atmospheric Science, Drexel University, Philadelphia, Pennsylvania 19104.
Biophys J. 1989 Oct;56(4):781-94. doi: 10.1016/S0006-3495(89)82725-0.
We have measured the forward and reverse rates of the allosteric transition of hemoglobin A with three CO molecules bound by using modulated excitation coupled with fluorescence quenching of the DPG analogue, PTS (8-hydroxy-1,3,6 pyrene trisulfonic acid). This dye is observed to bind to the T state with significantly larger affinity than to the R state, and thus provides an unequivocal marker for the molecule's conformational change. The allosteric rates obtained with the fluorescent dye (pH 7.0, bis-Tris buffer) are (3.4 +/- 1.0) x 10(3)s-1 for the R to T transition and (2.1 +/- 0.5) x 10(4)s-1 for the T to R transition. This gives an equilibrium constant L3 of 0.16 +/- 0.06. These results provide good agreement with modulated difference spectra calibrated from model compounds, arguing that there is little if any difference in the kinetics observed by the heme spectra and the kinetics of the full subunit motion. The equilibrium constant between structures (L3) is smaller in the absence of phosphates than observed in phosphate buffer (0.33). However, the rates of the allosteric transition increase in the absence of phosphates as compared with the corresponding rates in phosphate buffer of 1.0 x 10(3)s-1 and 3.0 x 10(3)s-1. The effects of inorganic phosphates on the equilibrium can be separated from the effects on kinetics. We find that phosphates also affect the dynamic behavior of hemoglobin, and the presence of 0.15 M phosphate can be viewed as raising the transition state energy between R and T conformations by approximately 0.5 kcal/mol exclusive of the T state stabilization. Dissociation constants for the dye were measured to be 104 +/- 25 microM for unligated T state and 930 +/- 300 microM for the fully ligated R state. The best fit equilibrium constant (125 +/- 40 microM) for three ligands bound does not differ significantly from that measured without ligands bound. Incidental to the measurement technique is the determination of the rates of binding and release of the dye. The association rate for binding to the T state is large, (at least 4 x 10(9) M-1 s-1) and may be diffusion limited, while the association and dissociation rates for R state binding, while not determined with precision, are clearly much smaller, of the scale of 10(5) M-1 s-1 for association.
我们通过调制激发结合DPG类似物PTS（8-羟基-1,3,6-芘三磺酸）的荧光猝灭，测量了结合三个CO分子的血红蛋白A变构转变的正向和反向速率。观察到这种染料与T态的结合亲和力明显大于与R态的结合亲和力，因此为分子的构象变化提供了明确的标记。用荧光染料（pH 7.0，双三羟甲基氨基甲烷缓冲液）获得的变构速率，R到T转变为(3.4±1.0)×10³ s⁻¹，T到R转变为(2.1±0.5)×10⁴ s⁻¹。这给出了平衡常数L3为0.16±0.06。这些结果与从模型化合物校准的调制差光谱很好地吻合，表明血红素光谱观察到的动力学与整个亚基运动的动力学几乎没有差异。在没有磷酸盐的情况下，结构之间的平衡常数（L3）比在磷酸盐缓冲液中观察到的要小（0.33）。然而，与磷酸盐缓冲液中1.0×10³ s⁻¹和3.0×10³ s⁻¹的相应速率相比，在没有磷酸盐的情况下变构转变速率增加。无机磷酸盐对平衡的影响可以与对动力学的影响分开。我们发现磷酸盐也会影响血红蛋白的动态行为，0.15 M磷酸盐的存在可被视为将R和T构象之间的过渡态能量提高约0.5千卡/摩尔，不包括T态的稳定化。未结合配体的T态染料解离常数测量为104±25 μM，完全结合配体的R态为930±300 μM。结合三个配体的最佳拟合平衡常数（125±40 μM）与未结合配体时测量的结果没有显著差异。测量技术附带的是染料结合和释放速率的测定。与T态结合的缔合速率很大（至少4×10⁹ M⁻¹ s⁻¹），可能受扩散限制，而R态结合的缔合和解离速率虽然没有精确测定，但明显小得多，缔合速率在10⁵ M⁻¹ s⁻¹范围内。
