Ivanova M, Jasuja R, Krasnosselskaia L, Josephs R, Wang Z, Ding M, Horiuchi K, Adachi K, Ferrone F A
Department of Physics, Drexel University, Philadelphia, PA 19104, USA.
J Mol Biol. 2001 Dec 7;314(4):851-61. doi: 10.1006/jmbi.2001.5163.
We have studied the self-assembly of Hemoglobin C-Harlem (HbC-Harlem), a double mutant of hemoglobin that possesses the beta6 Glu-->Val mutation of sickle hemoglobin (HbS) plus beta73 Asp-->Asn. By electron microscopy we find it forms crystals, rather than the wrapped multistranded fibers seen in HbS. Fourier transforms of the crystals yield unit cell parameters indistinguishable from crystals of HbS. Differential interference contrast (DIC) microscopy and birefringence also show crystal formation rather than the polymers or domains seen for HbS, while the growth patterns showed radiating crystal structures rather than simple linear crystalline forms. The solubility of the assembly was measured using a photolytic micromethod over a temperature range of 17-31 degrees C in 0.15 M phosphate buffer and found to be essentially the same as that of fibers of HbS. The assembly kinetics were observed by photolysis of the carbon monoxide derivative, and the mass of assembled hemoglobin was found to grow exponentially, with onset times that were stochastically distributed for small volumes. The stochastic onset of assembly showed strong concentration dependence, similar to but slightly greater than that seen in sickle hemoglobin nucleation. These observations suggest that like HbS, HbC-Harlem assembly proceeds by a homogeneous nucleation process, followed by heterogeneous nucleation. However, relative to HbS, both homogeneous and heterogeneous nucleation are suppressed by almost 11 orders of magnitude. The slowness of nucleation can be reconciled with the similarity of the solubility to HbS by an increase in contact energy coupled with a decrease in vibrational entropy recovered on assembly. This also explains the linearity of the double-strands, and agrees with the chemical nature of the structural replacement.
我们研究了血红蛋白C-哈勒姆（HbC-Harlem）的自组装过程，它是血红蛋白的一种双突变体，具有镰状血红蛋白（HbS）的β6谷氨酸→缬氨酸突变以及β73天冬氨酸→天冬酰胺突变。通过电子显微镜观察，我们发现它形成晶体，而非在HbS中所见的缠绕多链纤维。晶体的傅里叶变换得出的晶胞参数与HbS晶体的无法区分。微分干涉对比（DIC）显微镜和双折射也显示形成了晶体，而非HbS中所见的聚合物或结构域，同时生长模式呈现出放射状晶体结构，而非简单的线性晶体形式。在0.15 M磷酸盐缓冲液中，于17至31摄氏度的温度范围内，使用光解微方法测量了该组装体的溶解度，发现其与HbS纤维的溶解度基本相同。通过一氧化碳衍生物的光解观察组装动力学，发现组装的血红蛋白质量呈指数增长，对于小体积样品，起始时间呈随机分布。组装的随机起始显示出强烈的浓度依赖性，与镰状血红蛋白成核过程中所见相似，但略强。这些观察结果表明，与HbS一样，HbC-Harlem的组装通过均相成核过程进行，随后是异相成核。然而，相对于HbS，均相成核和异相成核都被抑制了近11个数量级。成核的缓慢可以通过组装时接触能的增加以及振动熵的减少来解释，这与HbS的溶解度相似性相一致。这也解释了双链的线性，并与结构替代的化学性质相符。
