具有谷胱甘肽过氧化物酶(GPx)活力的含硒四肽( SecRGD)

摘要

以精氨酸-甘氨酸-天冬氨酸(RGD)序列为基础,在N-端引入硒代半胱氨酸(Sec)设计了SecRGD序列模拟谷胱甘肽过氧化物酶(GPx),利用Fmoc固相合成法合成了secRGD.采用ESI-MS质谱和氢化物原子荧光光谱法对硒肽进行表征,采用酶偶联法进行GPx活力测定和酶动力学分析,用噻唑蓝(MTT)比色法评价了硒肽的抗氧化效果.结果表明,该硒肽的存在形式为SecRGD的二聚体.该硒肽具有GPx活力,其催化谷胱甘肽(GSH)还原H2O2的GPx活力为5.54 U/μmol,高于经典的GPx模拟物Ebselen.稳态动力学分析结果表明,该硒肽的催化机制为乒乓机制.该硒肽具有分子量小、易溶于水、毒性低及可有效保护Vero细胞免受氧化损伤的优点,具有作为抗氧化药物的应用前景.%Glutathione peroxidase ( GPx) is the most crucial antioxidant enzyme in a variety of organisms. Here, base on the Arg-Gly-Asp(RGD) , a tetrapeptide containing selenium(SeCRGD) was chemically synthesized. The existential state of the peptide was investigated by ESI-MS and hydride generation atom fluorescence spectral analysis. The GPx activities and the kinetics of the peptide were determined by the enzyme coupling method. The antioxidant effect of the peptide was evaluated based on MTT assay. The results showed that a kind of oxide form, SecRGD-dimer, was confirmed by ESI-MS and selenium content analysis. The peptide had GPx activity of 5. 54 U/μmol and more than that of Ebselen. As native enzyme, a ping-pong mechanism was observed in steady-state kinetics studies. Moreover, the novel selenopeptide occupied merits of small molecular weight, good solubility in water and low toxicity. Especially, the peptide was found that could protect Vero cells from the injury induced by H2O,. Thus, the novel selenopeptide possesses a good potential in medicinal application.