基于大环分子葫芦脲的鸟氨酸脱羧酶实时无标记活性分析方法的改进与应用

Optimization of a cucurbit[6]uril-based real-time label-free method for analyzing the activity of ornithine decarboxylase

摘要:

鸟氨酸脱羧酶 (Ornithine decarboxylase，ODC) 是多胺生物合成途径中的关键酶，其主要功能是催化鸟氨酸脱羧生成腐胺。在多种疾病和肿瘤细胞中，ODC的表达水平和催化活性都高于正常细胞，因此抑制ODC的活性是相关疾病预防和治疗的一个潜在途径。ODC抑制剂的发现和检验依赖于对其催化反应进程的监测，常采用的途径包括利用高效液相色谱法检测腐胺产量和利用同位素标记法检测二氧化碳的产量等。这些检测方法的繁琐操作和成本极大地限制了其应用，尤为突出的问题是这些方法很难实现高通量检测和实时检测。文中研究了基于大环分子葫芦[6]脲 (Cucurbit[6]uril，CB6) 与荧光染料DSMI (Trans-4-[4-(dimethylamino)styryl]-1-methylpyridinium iodide) 的ODC酶活实时无标记检测法，系统分析了其应用范围和局限性，并对其进行了优化。最后，利用优化后的方法实现了对不同机制ODC抑制剂的活性评价。

关键词:鸟氨酸脱羧酶，抑制剂，实时，无标记，酶活检测

Abstract:

Ornithine decarboxylase (ODC) is a key enzyme in the biosynthetic pathway of polyamines and catalyzes the decarboxylation of ornithine to produce putrescine. Inhibition of ODC activity is a potential approach for the prevention and treatment of many diseases including cancer, as the expression levels and the activities of ODC in many abnormal cells and tumor cells are generally higher than those of normal cells. The discovery and evaluation of ODC inhibitors rely on the monitoring of the reaction processes catalyzed by ODC. There are several commonly used methods for analyzing the activity of ODC, such as measuring the yield of putrescine by high performance liquid chromatography, or quantifying the yield of isotope labelled carbon dioxide. However, the cumbersome operation and cost of these assays, as well as the difficulty to achieve high-throughput and real-time detection, hampered their applications. In this work, we optimized a real-time label-free method for analyzing the activity of ODC based on the macromolecule cucurbit[6]uril (CB6) and a fluorescent dye, DSMI (trans-4-[4-(dimethylamino) styryl]-1-methylpyridinium iodide). Finally, the optimized method was used to determine the activities of different ODC inhibitors with different inhibition?mechanisms.

Key words:ornithine decarboxylase, inhibitor, real-time, label-free, enzyme activity analysis