Leicester Institute for Pharmaceutical Innovation, Faculty of Health and Life Sciences, De Montfort University, The Gateway, Leicester, LE1 9BH, UK.
Allied Health Sciences, Faculty of Health and Life Sciences, De Montfort University, The Gateway, Leicester, LE1 9BH, UK.
J Fluoresc. 2022 Mar;32(2):569-582. doi: 10.1007/s10895-021-02867-1. Epub 2022 Jan 10.
Fluorescently labelling proteins such as insulin have wide ranging applications in a pharmaceutical research and drug delivery. Human insulin (Actrapid®) was labelled with fluorescein isothiocyanate (FITC) and the synthesised conjugate identified using reverse phase high performance liquid chromatography (RP-HPLC) on a C18 column and a gradient method with mobile phase A containing 0.1% trifluoroacetic acid (TFA) in Millipore water and mobile phase B containing 90% Acetonitrile, 10% Millipore water and 0.1% TFA. Syntheses were carried out at varying reaction times between 4 and 20 h. Mono-labelled FITC-insulin conjugate was successfully synthesised with labelling at the B1 position on the insulin chain using a molar ratio of 2:1 (FITC:insulin) at a reaction time of 18 h and confirmed by electrospray mass spectroscopy. Reactions were studied across a pH range of 7-9.8 and the quantities switch from mono-labelled to di-labelled FITC-insulin conjugates at a reaction time of 2 h (2:1 molar ratio) at pH > 8. The conjugates isolated from the studies had biological activities in comparison to native insulin of 99.5% monoB1, 78% monoA1, 51% diA1B1 and 0.06% triA1B1B29 in HUVEC cells by examining AKT phosphorylation levels. MonoB1 FITC-insulin conjugate was also compared to native insulin by examining cell surface GLUT4 in C2C12 skeletal muscle cells. No significant difference in the cellular response was observed for monoB1 produced in-house compared to native insulin. Therefore mono-labelled FITC-insulin at the B1 position showed similar biological activity as native insulin and can potentially be used for future biomedical applications.
荧光标记蛋白质，如胰岛素，在药物研究和药物输送中有广泛的应用。人胰岛素（Actrapid®）用异硫氰酸荧光素（FITC）标记，并用 C18 柱反相高效液相色谱（RP-HPLC）和梯度方法，以含有 0.1%三氟乙酸（TFA）的 Millipore 水作为流动相 A 和含有 90%乙腈、10% Millipore 水和 0.1%TFA 的流动相 B 对合成的缀合物进行鉴定。在 4 至 20 小时的不同反应时间下进行合成。在 18 小时的反应时间下，使用摩尔比为 2:1（FITC:胰岛素），在胰岛素链的 B1 位置成功合成了单标记 FITC-胰岛素缀合物，并通过电喷雾质谱进行了确认。在 pH 7-9.8 的范围内进行了反应研究，在 pH>8 时，在 2 小时（2:1 摩尔比）的反应时间下，反应物从单标记物转变为双标记物 FITC-胰岛素缀合物。与天然胰岛素相比，从研究中分离出的缀合物在 HUVEC 细胞中具有生物活性，其 AKT 磷酸化水平分别为 99.5%的单 B1、78%的单 A1、51%的双 A1B1 和 0.06%的三 A1B1B29。在 C2C12 骨骼肌细胞中，通过检查细胞表面 GLUT4，还将单 B1 FITC-胰岛素缀合物与天然胰岛素进行了比较。与天然胰岛素相比，在细胞内产生的单 B1 没有观察到细胞反应的显著差异。因此，在 B1 位置的单标记 FITC-胰岛素具有与天然胰岛素相似的生物活性，可潜在地用于未来的生物医学应用。
