降纤酶的质量分析及其方法研究-药物分析专业论文.docx

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降纤酶的质量分析及其方法研究-药物分析专业论文.docx

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降纤酶的质量分析及其方法研究-药物分析专业论文

AbstractThepaperstudiedonthedefibrasecomingfromAgkistrodonacutusandAgkistrodonhalys.Reversedphase-highperformanceliquidchromatography(RP-HPLC)andisoelectricfocusing(IEF)wereusedtodeterminethepeptidemappingandisoelectricpoints.Sodiumdodecylsulfate-polyacrylamidegelelectrophoresis(SDS)wasperformedtoanalyzethemodeofactionofdifferentdefibrasesonfibrinogen.RP-HPLCwasusedtotestthepurityofdefibraseanddeterminethecontentindefibraseforinjection.Themethodoffibrinogencoagulationwasperformedtodeterminetheactivity.SDSandmatrix-assistedlaserdesorption/ionization-timeofflight-massspectrometry(MALDI-TOF-MS)wereusedtodeterminethemolecularweight.Phenylisothiocyanate(PITC)-pre-columnderivatizationandthemethodofEdmandegradationwereperformedtodeterminethecompositionofaminoacidsandtheN-terminalsequences.N-linkedoligosaccharidespectrumswereobtainedbynormalphase-highperformanceliquidchromatography(NP-HPLC).Monosaccharidecompositiondeterminationofdefibrasewasperformedbyhighperformanceanion-exchangechromatographywithpulsedamperometricdetection(HPAEC-PAD).Meanwhile,defibrasesforinjectionwhichcamefromdifferentdomesticpharmaceutical factorieswere analyzed,andrelationshipbetween thecontentandactivityofdefibrasewasstudied.Wegavesomesuggestionsfortherevisementofcurrentstandards.ItindicatedthattheresultsinRP-HPLCpeptidemappingwerequitedifferentbetweendefibrasesfromAgkistrodonacutusandAgkistrodonhalysandtheirisoelectricpointswere3.5and4.5-8.0,respectively.DefibrasecomingfromAgkistrodonacutus actedontheαchainoffibrinogen,butdefibrasefromAgkistrodonhalysactedonbothoftheαandtheβchains.RP-HPLCcanbeusedtodeterminethepurityandthecontentofdefibraseininjections.Todeterminetheactivityof defibrase,thesubstitutionof clottingdeviceforvisualmeasurementwastotallyfeasible.TheresultsofMALDI-TOF-MSanalysisshowedthatthemolecularweightofdefibrasefromAgkistrodonacutuswas33363.95whiletheonefromAgkistrodonhalyswas33633.75.Bothof thedifferentcomingdefibraseshadthesamemolecularweightsof40kDbySDS.Theircompositionofaminoacid