BTx PharmSci Pharmaceutical R&D , Pfizer, Inc. , One Burtt Road , Andover 01810 , Massachusetts , United States.
Boreskov Institute of Catalysis, Siberian Branch of the RAS , Lavrentieva Avenue, 5 , Novosibirsk 630090 , Russia.
J Phys Chem B. 2019 Jul 11;123(27):5690-5699. doi: 10.1021/acs.jpcb.9b02443. Epub 2019 Jul 1.
Protein/ice interactions are investigated by a novel method based on measuring the characteristic features of X-ray diffraction (XRD) patterns of hexagonal ice (Ih). Aqueous solutions of four proteins and other solutes are studied using high-resolution synchrotron XRD. Two pharmaceutical proteins, recombinant human albumin and monoclonal antibody (both at 100 mg/mL), have a pronounced effect on the properties of ice crystals, reducing the size of the Ih crystalline domains and increasing the microstrain. Lysozyme (100 mg/mL) and an antifreeze protein (1 mg/mL) have much weaker impact on Ih. Neither of the proteins studied exhibit preferred interactions with specific crystalline faces of Ih. It is proposed that the pharmaceutical proteins interact with ice crystals indirectly by accumulating in the quasi-liquid layer next to ice crystallization front, rather than directly, via a sorption on ice crystals. This is the first report, to the best of our knowledge, of major difference in the protein/ice interaction between non-antifreeze proteins. Another important finding is a detection of a second (minor) population of ice crystals, which is tentatively identified as a high-pressure form of ice, possibly IceIII or IceIX. This finding highlights a potential role of mechanical stresses in freeze-induced destabilization of proteins.
蛋白质/冰相互作用是通过一种基于测量六方冰（Ih）的 X 射线衍射（XRD）图谱特征的新方法来研究的。使用高分辨率同步加速器 XRD 研究了四种蛋白质和其他溶质的水溶液。两种药物蛋白，重组人白蛋白和单克隆抗体（均为 100mg/mL），对冰晶的性质有明显影响，减小了 Ih 结晶区的大小并增加了微应变。溶菌酶（100mg/mL）和抗冻蛋白（1mg/mL）对 Ih 的影响要弱得多。研究中没有一种蛋白质表现出与 Ih 特定晶面的优先相互作用。据我们所知，这是首次报道非抗冻蛋白与冰的相互作用存在显著差异。另一个重要的发现是检测到第二种（次要）冰晶群体，其暂定为冰的高压形式，可能是 IceIII 或 IceIX。这一发现突出了机械应力在冷冻诱导蛋白质失稳中的潜在作用。
