Department of Physics, Drexel University, Philadelphia, Pennsylvania, USA.
Biophys J. 2011 Aug 17;101(4):885-91. doi: 10.1016/j.bpj.2011.05.064.
The measurement of polymer growth is an essential element in characterization of assembly. We have developed a precise method of measuring the growth of sickle hemoglobin polymers by observing the time required for polymers to traverse a photolytically produced channel between a region in which polymers are created and a detection region. The presence of the polymer is functionally detected by observing its ability to create new polymers through the well-established process of heterogeneous nucleation. Using this method, we have determined the rate constants for monomer addition to and release from polymer ends, as well as their temperature dependences. At 25°C we find k(+) = 84 ± 2 mM⁻¹ s⁻¹ and k(-) = 790 ± 80 molecules/s from each end. These numbers are in accord with differential interference contrast measurements, and their ratio gives a solubility measured on individual fibers. The single-fiber solubility agrees with that measured in sedimentation experiments. The concentration dependence of the monomer addition rate is consistent with monomer addition, but not oligomer addition, to growing polymers. The concentration dependence suggests the presence of an activation enthalpy barrier, and the rate of monomer addition is not diffusion-limited. Analysis of the temperature dependence of the monomer addition rate reveals an apparent activation energy of 9.1 ± 0.6 kcal/mol.
聚合物生长的测量是组装特性分析的基本要素。我们开发了一种精确的方法，通过观察聚合物在光解产生的通道中从聚合物生成区域到检测区域所需的时间来测量镰状血红蛋白聚合物的生长。聚合物的存在通过观察其通过成熟的异质成核过程形成新聚合物的能力来功能检测。使用这种方法，我们确定了单体添加到聚合物末端和从聚合物末端释放的速率常数及其温度依赖性。在 25°C 下，我们从每个末端发现 k(+) = 84 ± 2 mM⁻¹ s⁻¹ 和 k(-) = 790 ± 80 个分子/s。这些数字与微分干涉对比测量一致，并且它们的比值给出了在单个纤维上测量的溶解度。单纤维溶解度与沉降实验中测量的溶解度一致。单体添加速率的浓度依赖性与单体向生长聚合物的添加一致，但与低聚物的添加不一致。浓度依赖性表明存在活化焓垒，并且单体添加的速率不是扩散限制的。单体添加速率的温度依赖性分析揭示了 9.1 ± 0.6 kcal/mol 的表观活化能。
