Weiss William F, Hodgdon Travis K, Kaler Eric W, Lenhoff Abraham M, Roberts Christopher J
Department of Chemical Engineering, University of Delaware, Newark, Delaware, USA.
Biophys J. 2007 Dec 15;93(12):4392-403. doi: 10.1529/biophysj.107.112102. Epub 2007 Aug 17.
Thermally induced aggregates of alpha-chymotrypsinogen A and bovine granulocyte-colony stimulating factor in acidic solutions were characterized by a combination of static and dynamic light scattering, spectroscopy, transmission electron microscopy, and monomer loss kinetics. The resulting soluble, high-molecular weight aggregates (approximately 10(3)-10(5) kDa) are linear, semiflexible polymer chains that do not appreciably associate with one another under the conditions at which they were formed, with classic power-law scaling of the radius of gyration and hydrodynamic radius with weight-average molecular weight (M(w)). Aggregates in both systems are composed of nonnative monomers with elevated levels of beta-sheet secondary structure, and bind thioflavine T. In general, the aggregate size distributions showed low polydispersity by light scattering. Together with the inverse scaling of M(w) with protein concentration, the results clearly indicate that aggregation proceeds via nucleated (chain) polymerization. For alpha-chymotrypsinogen A, the scaling behavior is combined with the kinetics of aggregation to deduce separate values for the characteristic timescales for nucleation (tau(n)) and growth (tau(g)), as well as the stoichiometry of the nucleus (x). The analysis illustrates a general procedure to noninvasively and quantitatively determine tau(n), tau(g), and x for soluble (chain polymer) aggregates, as well as the relationship between tau(n)/tau(g) and aggregate M(w).
通过静态和动态光散射、光谱学、透射电子显微镜以及单体损失动力学相结合的方法，对酸性溶液中热诱导的α-胰凝乳蛋白酶原A和牛粒细胞集落刺激因子聚集体进行了表征。所形成的可溶性高分子量聚集体（约10³ - 10⁵ kDa）是线性的半柔性聚合物链，在形成条件下它们彼此之间没有明显的缔合，其回转半径和流体力学半径与重均分子量（M(w)）呈现经典的幂律标度关系。两个体系中的聚集体均由具有升高的β-折叠二级结构水平的非天然单体组成，并与硫黄素T结合。一般来说，通过光散射得到的聚集体尺寸分布显示出低多分散性。结合M(w)与蛋白质浓度的反比标度关系，结果清楚地表明聚集是通过成核（链）聚合进行的。对于α-胰凝乳蛋白酶原A，将标度行为与聚集动力学相结合，推导出成核（τ(n)）和生长（τ(g)）的特征时间尺度的单独值，以及核的化学计量比（x）。该分析说明了一种通用方法，可用于无创且定量地确定可溶性（链聚合物）聚集体的τ(n)、τ(g)和x，以及τ(n)/τ(g)与聚集体M(w)之间的关系。
