Mehta Shyam B, Bee Jared S, Randolph Theodore W, Carpenter John F
Department of Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus , Aurora, Colorado 80045, United States.
Biochemistry. 2014 May 27;53(20):3367-77. doi: 10.1021/bi5002163. Epub 2014 May 15.
We have examined the effect of incubating a monoclonal antibody (mAb) in low (0-2.0 M) concentrations of guanidine hydrochloride (GdnHCl) on the protein's conformation and aggregation during isothermal incubation. In GdnHCl solutions at concentrations from 1.2 to 1.6 M, the mAb was partially unfolded. As demonstrated by fluorescence and circular dichroism spectroscopy, the partially unfolded state of the antibody had perturbed tertiary structure but retained native secondary structure. Furthermore, partial unfolding of the antibody was documented by analytical ultracentrifugation, dynamic light scattering, and limited proteolysis. Subsequent aggregation of the antibody was characterized using size-exclusion chromatography, analytical ultracentrifugation, and dynamic light scattering. Over the entire concentration range (0-2.0 M) of GdnHCl, protein-protein interactions were attractive, as quantified by negative osmotic second virial coefficients measured with static light scattering. However, during isothermal incubation at 37 °C, the aggregation of the antibody was detected only in solutions that induced partial unfolding. Differential scanning calorimetry studies showed that the antibody's CH2 domains were unfolded in antibody molecules that had been incubated in 1.2 M and higher concentrations of GdnHCl. These results suggest that unfolding of the CH2 domains leads to aggregation.
我们研究了在低浓度(0 - 2.0 M)盐酸胍(GdnHCl)中孵育单克隆抗体(mAb)对等温孵育过程中蛋白质构象和聚集的影响。在浓度为1.2至1.6 M的GdnHCl溶液中,mAb发生了部分展开。荧光光谱和圆二色光谱表明,抗体的部分展开状态扰乱了三级结构,但保留了天然二级结构。此外,通过分析超速离心、动态光散射和有限蛋白酶解证明了抗体的部分展开。随后使用尺寸排阻色谱、分析超速离心和动态光散射对抗体的聚集进行了表征。在GdnHCl的整个浓度范围(0 - 2.0 M)内,通过静态光散射测量的负渗透第二维里系数量化表明,蛋白质 - 蛋白质相互作用是吸引性的。然而,在37°C等温孵育期间,仅在诱导部分展开的溶液中检测到抗体的聚集。差示扫描量热法研究表明,在1.2 M及更高浓度GdnHCl中孵育的抗体分子中,抗体的CH2结构域发生了展开。这些结果表明,CH2结构域的展开导致了聚集。