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本文设计可活化细胞穿膜肽(activatable cell-penetrating peptide,ACPP),对合成的ACPP进行酶解研究。ACPP由3部分连接而成,寡聚阴离子序列肽-基质金属蛋白酶(matrix metalloproteinase,MMP)的底物肽-寡聚阳离子序列肽。采用反相高效液相色谱(reversed-phase high performance liquid chromatography,RP-HPLC)监测37℃条件下IV型胶原酶(含MMP-2和MMP-9)对ACPP的酶解过程。收集酶解成分进行基质辅助激光解析电离飞行时间质谱(matrix assisted laser desorption ionization orthogonal time of flight mass spectrometry,MALDIO-TOF-MS)检测,比对推测酶解碎片的序列。结果表明,ACPP可被IV型胶原酶裂解,释放出寡聚阳离子序列肽,即细胞穿膜肽(cell-penetrating peptide,CPP),ACPP裂解一半的时间约为4h。酶解发生于目标位点,但不排除ACPP裂解后肽段的再次断裂。
In this paper, we designed an activatable cell-penetrating peptide (ACPP) to study the enzymatic hydrolysis of ACPP. ACPP is composed of three parts, the oligopeptide sequence of matrix metalloproteinase (MMP). The enzymatic hydrolysis of ACPP by type IV collagenase (including MMP-2 and MMP-9) at 37 ℃ was monitored by reversed-phase high performance liquid chromatography (RP-HPLC) The enzymatic components were collected and analyzed by matrix assisted laser desorption ionization (orthogonal time-of-flight mass spectrometry, MALDIO-TOF-MS). The results showed that ACPP was cleaved by type IV collagenase and released an oligopeptide sequence called cell-penetrating peptide (CPP). The cleavage time of ACPP was about 4 h. Enzymolysis occurs at the target site, but does not rule out the cleavage of ACPP cleavage of the peptide again.