Study on the Detection of MMP-2，-7，-9，and -12 Enzymatic Activity Using CEACAM1-Derived Fluorescent Peptide Substrate Site 84
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摘要:
目的 探索CEACAM1 源性多肽合成的荧光底物Site 84 对MMP-2，-7，-9，-12 酶活性的检测，并进一步探索荧光多肽底物Site 84在区分同属于MMPs明胶酶谱中MMP-2和MMP-9的应用。 方法 采用荧光酶学法观察荧光多肽底物Site 84 对MMP-2，-7，-9，-12 酶活性的检测；进一步观察底物Site 84对明胶酶谱中MMP-2和MMP-9酶活性的敏感性和特异性；获得底物Site 84与MMP-2的酶促反应的动力学参数Km、Kcat。 结果 以Site 84 为底物，获得MMP-12、-7、-2 的酶活力曲线，但未获得MMP-9的酶活力曲线；此外，以Site 84 为底物，可特异性检测明胶酶谱中MMP-2 的酶活性，其可检测低浓度（0.6 μM）MMP-2 的酶活性，然而对高浓度（6 μM）MMP-9 未见明显酶促反应；最后，MMP-2 和底物 Site 84的酶促反应动力学参数：Km = 315 μM，Kcat/Km = 2565 / MS。结论 以CEACAM1源性底物 Site 84作为新型的荧光多肽底物，可获得MMP-12、-7、-2 的酶活力曲线，且可特异性检测MMPs明胶酶谱中MMP-2的酶活性。 Abstract:Objective To explore the the detection of MMP-2, -7, -9, and -12 enzymatic activity using the CEACAM1-derived fluorescent peptide substrate Site 84, investigating the application of substrate Site 84 to distinguishing between MMP-2 and MMP-9 in the gelatinase spectrum of MMPs. Methods The fluorescent enzymatic method was employed to observe the detection of MMP-2, -7, -9, and -12 enzymatic activity using substrate Site 84; further observations were made on the sensitivity and specificity of substrate Site 84 to enzymatic activity of MMP-2 and MMP-9 within the gelatinase spectrum; the kinetic parameters （Km and Kcat） of the enzymatic reaction between substrate Site 84 and MMP-2 were obtained. Results Using Site 84 as a substrate, enzymic kinetics curves for MMP-12, -7, -2 were obtained, but no enzymatic activity curve for MMP-9 was observed. Furthermore, Site 84 specifically detected the enzymatic activity of MMP-2 within the gelatinase spectrum, capable of detecting low concentration （0.6 μM） of MMP-2 enzymatic activity, but no obvious enzymatic reaction was observed for high concentration （6 μM） of MMP-9; the kinetics parameters for the enzymatic reaction between Site 84 and MMP-2 were Km = 315 μM, Kcat/Km = 2565 /MS.Conclusion The CEACAM1-derived substrate Site 84 serves as a novel fluorescent peptide substrate, enabling the acquisition of enzymatic activity curves for MMP-12, -7 and -2, and specifically detecting the enzymatic activity of MMP-2 within the MMP gelatinase spectrum. -
Key words:
- Site 84 /
- Fluorescent polypeptide substrate /
- Enzymatic activity /
- Gelatinase /
- CEACAM1-derived.
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图 1 Site84的高效液相色谱（HPLC）分析图和质谱分析图
A：HPLC纯化图，波峰即为Site 84；B：图A中波峰的二次质谱图。
Figure 1. HPLC analysis and mass spectrometry analysis of Site 84
图 2 rhMMPs酶活性位点滴定
A：MMP-12的酶活性浓度；B：MMP-7的酶活性浓度；C：MMP-2的酶活性浓度；D：MMP-9的酶活性浓度。
Figure 2. Titration of enzymatic activity sites of rhMMPs
图 3 MMP-2/-7/-9/-12 与荧光多肽底物Site 84的酶促反应
Figure 3. Enzymatic reactions of MMP-2/-7/-9/-12 with fluorescent peptide substrate Site 84
图 4 明胶酶（MMP-2 及 MMP-9）与荧光多肽底物 Site 84的酶促反应
Figure 4. Enzymatic reactions of gelatinases （MMP-2 and MMP-9） with fluorescent peptide substrate Site 84
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