CEACAM1源性荧光多肽底物Site 84对MMP-2、-7、-9、-12酶活性检测的研究  

Study on the Detection of MMP-2,-7,-9,and-12 Enzymatic Activity Using CEACAM1-Derived Fluorescent Peptide Substrate Site 84

作　　者：万雯[1] 叶雨佳 杨晓娜 杨理宏 王华炜[1] 董玲 陈丽星[2] 孟照辉[1] WAN Wen;YE Yujia;YANG Xiaona;YANG Lihong;WANG Huawei;DONG Ling;CHEN Lixing;MENG Zhaohui(Department 1 of Cardiology,The First Affiliated Hospital of Kunming Medical University,Kunming Yunnan 650032;Department 3 of Cardiology,The First Affiliated Hospital of Kunming Medical University,Kunming Yunnan 650032,China)

机构地区：[1]昆明医科大学第一附属医院心脏内一科,云南昆明650032 [2]昆明医科大学第一附属医院心脏内三科,云南昆明650032

出　　处：《昆明医科大学学报》2025年第2期9-16,共8页Journal of Kunming Medical University

基　　金：国家自然科学基金(82360077);云南省基础研究计划昆医联合专项-面上项目(202201AY070001-082);云南省基础研究计划青年项目(202201AU070063);昆明医科大学第一附属医院“535”高层次人才(2024535Q03)。

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(0.6μM)MMP-2的酶活性,然而对高浓度(6μM)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=315μM,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。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(0.6μM)of MMP-2 enzymatic activity,but no obvious enzymatic reaction was observed for high concentration(6μM)of MMP-9;the kinetics parameters for the enzymatic reaction between Site 84 and MMP-2 were Km=315μM,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.

关 键 词：Site 84 荧光多肽底物 酶活性 明胶酶 CEACAM1源性 

分 类 号：R541.4[医药卫生—心血管疾病]