Department of Pharmacy, University of Copenhagen, Copenhagen, Denmark; Novo Nordisk A/S, Måløv, Denmark; Department of Pharmaceutical Sciences, University of Colorado-Denver, Aurora, Colorado 80045.
KBI Biopharma, Boulder, Colorado 80301.
J Pharm Sci. 2019 Sep;108(9):2871-2879. doi: 10.1016/j.xphs.2019.04.018. Epub 2019 Apr 23.
Protein fibrils are of great interest due to their involvement in several pathologies and their roles in the degradation of many therapeutic protein products. Fibrils share highly similar secondary structural motifs across different proteins and applied stress conditions. However, fibril morphology differs according to the surrounding conditions, with aromatic and hydrophobic amino acids playing important roles in mature fibril formation. In this study, we use Raman microscopy, by means of the aromatic amino acids in insulin molecules as markers, to probe for tertiary structure differences within fibrils. We compared 2 different fibril types, linear fibril bundles and spherulites. Generation of linear fibril bundles was undertaken in an acetic acid-containing formulation, whereas spherulites were generated in a hydrochloric acid-containing formulation. The Raman intensities of tyrosine and phenylalanine side chains suggest that there are significant differences between the fibril bundles. The findings suggest that the insulin components of the fibril strands are not arranged identically in the 2 fibril types and that this gives rise to differences in their tertiary structures. Overall, the work indicates that the physicochemical properties of fibril structures can be altered by changing the formulation and that these alterations can be monitored by Raman spectroscopy.
蛋白质纤维因其在多种病理学中的作用以及在许多治疗性蛋白质产品降解中的作用而备受关注。纤维在不同的蛋白质和应用的压力条件下具有高度相似的二级结构模体。然而，根据周围条件的不同，纤维形态也会有所不同，芳香族和疏水性氨基酸在成熟纤维的形成中起着重要作用。在这项研究中，我们使用拉曼显微镜，通过胰岛素分子中的芳香族氨基酸作为标记物，来探测纤维内的三级结构差异。我们比较了 2 种不同的纤维类型，线性纤维束和球晶。线性纤维束是在含有乙酸的配方中生成的，而球晶是在含有盐酸的配方中生成的。酪氨酸和苯丙氨酸侧链的拉曼强度表明，纤维束之间存在显著差异。研究结果表明，纤维链中的胰岛素成分在这 2 种纤维类型中的排列方式不同，这导致了它们的三级结构的差异。总的来说，这项工作表明可以通过改变配方来改变纤维结构的物理化学性质，并且可以通过拉曼光谱来监测这些变化。
