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1、·1048·药学学报ActaPharmaceuticaSinica2010,45(8):1048−1051可活化细胞穿膜肽的设计与酶解研究*王鹏程,齐宪荣(北京大学药学院药剂学研究室,北京100191)摘要:本文设计可活化细胞穿膜肽(activatablecell-penetratingpeptide,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(matrixmetalloproteinase,MMP)的底物肽-寡聚阳离子序列肽。采用反相高效液相色谱(reversed-phasehigh
2、performanceliquidchromatography,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(matrixassistedlaserdesorptionionizationorthogonaltimeofflightmassspectrometry,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-penetratingpeptide,
3、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。关键词:细胞穿膜肽;酶解;基质金属蛋白酶;反相高效液相色谱;基质辅助激光解析电离飞行时间质谱中图分类号:R943文献标识码:A文章编号:0513-4870(2010)08-1048-04Thedesignandenzymatichydrolysisofactivatablecell-penetratingpeptide*WANGPeng-cheng,QIXian-rong(SchoolofPharmaceuticalSciences,Pekin
4、gUniversity,Beijing100191,China)Abstract:Thepaperisaimedtostudytheenzymatichydrolysisoftheactivatablecell-penetratingpeptide(ACPP)thatwasdesignedandsynthesized.TheACPPwascomposedofthreeparts,polyanionicsequencepeptide,peptidesequencethatspecificallycleavedbymatrixmetallopro
5、teinase(MMP)andcellpenetratingpeptide(CPP).TheACPPwashydrolyzedbytypeIVcollagenase(MMP-2/9)undertheconditionof37℃andwasmonitoredbyreversed-phasehighperformanceliquidchromatography(RP-HPLC).Theeffluxofpeakwascollectedanddetectedbymatrixassistedlaserdesorptionionizationorthog
6、onaltimeofflightmassspectrometry(MALDIO-TOF-MS)tospeculatethesequencesofthepeptidefragments.TheresultsindicatedthattheACPPcouldbecleavedbytypeIVcollagenaseattargetsiteaspredicted,releasedCPP.Thehalflifeofthecleavagewasabout4h.Meanwhile,thepeptidefragmentsmaybecleavedagainat
7、othersitesbytypeIVcollagenase.Keywords:cell-penetratingpeptide;enzymatichydrolysis;matrixmetalloproteinase;reversed-phasehighperformanceliquidchromatography;matrixassistedlaserdesorptionionizationorthogonaltimeofflightmassspectrometry收稿日期:2010-02-25.基金项目:国家自然科学基金资助项目(307726
8、65,30970785).*通讯作者Tel/Fax:86-10-82801584,E-mail:qixr2001@yahoo.com.cn王鹏程等:可活化细胞穿膜肽
