School of Chemistry and Chemical Engineering, Huazhong University of Science and Technology, Wuhan, China.
J Enzyme Inhib Med Chem. 2011 Apr;26(2):181-7. doi: 10.3109/14756366.2010.487485.
A novel high-performance liquid chromatography (HPLC) method based on the internal standard method was established for assaying the tumour necrosis factor-α converting enzyme (TACE) activity and matrix metalloprotease-9 (MMP-9) activity, and was used to evaluate the inhibitive effectiveness of inhibitors to TACE and MMP-9. In the assay method for TACE and MMP-9, peptides labelled with the ultraviolet group-Dpa were used as substrates. Alanine-Dpa was synthesised and was used as the internal standard for quantitative analysis. After the peptide substrates were hydrolysed by TACE (MMP-9) for 15 min (25 min) at 37 °C, the amount of remaining substrates were determined by reversed-phased HPLC with UV detection at 353 nm. The relative peak area of the substrate was linearly dependent on the substrate concentration. This method was then applied to determine the 50% inhibitory concentration (IC₅₀) of GM6001 and inhibitor A for both TACE and MMP-9.
建立了一种基于内标法的新型高效液相色谱（HPLC）法，用于测定肿瘤坏死因子-α转化酶（TACE）活性和基质金属蛋白酶-9（MMP-9）活性，并用于评估 TACE 和 MMP-9 抑制剂的抑制效果。在 TACE 和 MMP-9 的测定方法中，使用标记有紫外基团-Dpa 的肽作为底物。合成了丙氨酸-Dpa 并用作定量分析的内标。肽底物在 37°C 下被 TACE（MMP-9）水解 15 分钟（25 分钟）后，通过反相 HPLC 用 UV 检测在 353nm 处测定剩余底物的量。底物的相对峰面积与底物浓度呈线性依赖关系。然后，该方法用于测定 GM6001 和抑制剂 A 对 TACE 和 MMP-9 的 50%抑制浓度（IC₅₀）。
