Division of Drug Delivery Technology Leiden/Amsterdam Center for Drug Research (LACDR), Leiden University, P.O. Box 9502, 2300, Leiden, The Netherlands.
Pharm Res. 2012 Aug;29(8):2276-93. doi: 10.1007/s11095-012-0755-z. Epub 2012 May 10.
To elucidate the chemical modifications in covalent aggregates of recombinant human insulin induced by metal catalyzed oxidation (MCO).
Insulin was exposed for 3 h at room temperature to the oxidative system copper(II)/ascorbate. Chemical derivatization with 4-(aminomethyl) benzenesulfonic acid (ABS) was performed to detect 3,4-dihydroxyphenylalanine (DOPA) formation. Electrospray ionization-mass spectrometry (ESI-MS) was employed to localize the amino acids targeted by oxidation and the cross-links involved in insulin aggregation. Oxidation at different pH and temperature was monitored with size exclusion chromatography (SEC) and ESI-MS analysis to further investigate the chemical mechanism(s), to estimate the aggregates content and to quantify DOPA in aggregated insulin.
The results implicate the formation of DOPA and 2-amino-3-(3,4-dioxocyclohexa-1,5-dien-1-yl) propanoic acid (DOCH), followed by Michael addition, as responsible for new cross-links resulting in covalent aggregation of insulin during MCO. Michael addition products were detected between DOCH at positions B16, B26, A14, and A19, and free amino groups of the N-terminal amino acids Phe B1 and Gly A1, and side chains of Lys B29, His B5 and His B10. Fragments originating from peptide bond hydrolysis were also detected.
MCO of insulin leads to covalent aggregation through cross-linking via Michael addition to tyrosine oxidation products.
阐明金属催化氧化（MCO）诱导的重组人胰岛素共价聚集体中的化学修饰。
胰岛素在室温下暴露于氧化体系铜（II）/抗坏血酸中 3 小时。用 4-（氨甲基）苯磺酸（ABS）进行化学衍生化，以检测 3,4-二羟基苯丙氨酸（DOPA）的形成。采用电喷雾电离质谱（ESI-MS）定位氧化作用所针对的氨基酸和胰岛素聚集所涉及的交联。通过尺寸排阻色谱（SEC）和 ESI-MS 分析监测不同 pH 值和温度下的氧化作用，以进一步研究化学机制，估计聚合体含量，并量化聚集胰岛素中的 DOPA。
结果表明，DOPA 和 2-氨基-3-（3,4-二氧环己-1,5-二烯-1-基）丙酸（DOCH）的形成，随后是迈克尔加成，是导致 MCO 过程中胰岛素发生新的交联并导致其共价聚合的原因。在 DOCH 位置 B16、B26、A14 和 A19 与 N 末端氨基酸 Phe B1 和 Gly A1 的游离氨基以及 Lys B29、His B5 和 His B10 的侧链之间检测到迈克尔加成产物。还检测到源自肽键水解的片段。
胰岛素的 MCO 通过酪氨酸氧化产物的迈克尔加成交联导致共价聚合。
