Nielsen L, Frokjaer S, Carpenter J F, Brange J
Department of Pharmaceutics, The Royal Danish School of Pharmacy, 2100 Copenhagen, Denmark.
J Pharm Sci. 2001 Jan;90(1):29-37. doi: 10.1002/1520-6017(200101)90:1<29::aid-jps4>3.0.co;2-4.
Fibril formation (aggregation) of insulin was investigated in acid media by visual inspection, transmission electron microscopy (TEM), and Fourier transform infrared (FTIR) spectroscopy. Insulin fibrillated faster in hydrochloric acid than in acetic acid at elevated temperatures, whereas the fibrillation tendencies were reversed at ambient temperatures. Electron micrographs showed that bovine insulin fibrils consisted of long fibers with a diameter of 5 to 10 nm and lengths of several microns. The fibrils appeared either as helical filaments (in hydrochloric acid) or arranged laterally in bundles (in acetic acid, NaCl). Freeze-thawing cycles broke the fibrils into shorter segments. FTIR spectroscopy showed that the native secondary structure of insulin was identical in hydrochloric acid and acetic acid, whereas the secondary structure of fibrils formed in hydrochloric acid was different from that formed in acetic acid. Fibrils of bovine insulin prepared by heating or agitating an acid solution of insulin showed an increased content of beta-sheet (mostly intermolecular) and a decrease in the intensity of the alpha-helix band. In hydrochloric acid, the frequencies of the beta-sheet bands depended on whether the fibrillation was induced by heating or agitation. This difference was not seen in acetic acid. Freeze-thawing cycles of the fibrils in hydrochloric acid caused an increase in the intensity of the band at 1635 cm(-1) concomitant with reduction of the band at 1622 cm(-1). The results showed that the structure of insulin fibrils is highly dependent on the composition of the acid media and on the treatment.
通过目视检查、透射电子显微镜（TEM）和傅里叶变换红外（FTIR）光谱法，在酸性介质中研究了胰岛素的原纤维形成（聚集）过程。在升高的温度下，胰岛素在盐酸中比在乙酸中更快地形成原纤维，而在环境温度下，原纤维形成的趋势则相反。电子显微镜照片显示，牛胰岛素原纤维由直径为5至10纳米、长度为几微米的长纤维组成。原纤维呈现为螺旋状细丝（在盐酸中）或横向排列成束（在乙酸、氯化钠中）。冻融循环将原纤维分解成更短的片段。FTIR光谱表明，胰岛素的天然二级结构在盐酸和乙酸中是相同的，而在盐酸中形成的原纤维的二级结构与在乙酸中形成的不同。通过加热或搅拌胰岛素的酸性溶液制备的牛胰岛素原纤维显示β-折叠（主要是分子间的）含量增加，α-螺旋带的强度降低。在盐酸中，β-折叠带的频率取决于原纤维形成是由加热还是搅拌诱导的。在乙酸中未观察到这种差异。盐酸中原纤维的冻融循环导致1635 cm⁻¹处的带强度增加，同时1622 cm⁻¹处的带强度降低。结果表明，胰岛素原纤维的结构高度依赖于酸性介质的组成和处理方式。
