实验六谷丙转氨酶活性测定

实验六

观察血清中谷丙转氨酶的活力变化

Experiment6ObservingEnzymeActivityChangesofAlanineAminotransferaseinSerumSampleExperimentalpurposeKnowthesignificanceofactivityofserumALT(GPT).MastertheassayoftheactivityofALT.Experimentaltips1.transaminationaminotransferase2.aminotransferase，transaminase

Definition:

Anenzymewhichcouldtransferamino-groupofanaminoacidtoanothercarbonylgroupofaketonecompoud;Products:anewaminoacidandanewketoneNofreeaminogroup!Cofactor:pyridoxalphosphate3.glutaimc-pyruvictransminaseGPT(谷丙转氨酶)oralanineaminotransferaseALT(丙氨酸转氨酶)pyruvicacidglutamicacidketoglutaratealanine4.glutamicoxaloacetictransaminaseGOT(谷草转氨酶)

oraspartatetransaminase,AST（天冬氨酸转氨酶）glutamicacidoxaloaceticacidketoglutarateaspartate5.clinicalsignificanceALTandASTcouldbeusedtoindicatethepathologicalchangesofliverandheart;TheactivitiesofALTandASTinliverandheartarehigherthanothertissues;TheactivityofALTinliverislowerthan40units.ExperimentalPrincipleSUBSTRACTphenylhydrazone2,4-dinitratephenylhydrazinealanineα-ketoglutarateRedbrownpyruvicacidpyruvicacidglutamicacidactivityofSALT：oneSALTunitisdeifinedasthattheenzymaticamountwhichproduced2.5microgramme(μg)pyruvicacidundertheconditionofthat1mlserumreactwithsubstanceat37℃for30minutes.ActivityofSALTThenumberofμgofmeasuretube—

thatofconctrol2.5×0.1

（U）

=Experimentalprocedure1.

StandardcurveRegentsTube012345STDpyruvicacidsolution，mlSALTsubstratesolution，mlPhosphatebuffer,PBS，ml0.000.500.100.050.450.100.100.400.100.150.350.100.200.300.100.250.250.1037℃

for10min2,4-dinitratephenylhydrazine,ml0.500.500.500.500.500.5037℃

for20minNaOHsolution,ml55555510minatroomtemperatureDeterminetheabsorptionat520nm

tubesAddstandardpyrivicacidsolution,SALTsubstratesolutionandphosphatebufferWaterbathingat37℃for10’Add2,4-dinitratephenylhydrazineandwaterbathat37℃for20’AddNaOHsolutionandkeepinroomtemperaturefor10’Assayat520nm

2．Determinationoftheactivityof

SALTRegentsAssaytubeControltubeSerum,mlSALT

substratesolution,ml0.10.50.1____37℃

for30min2,4-dinitratephenylhydrazine,mlSALTsubstratesolution,

ml0.5____0.50.537℃

for20min0.4mol/LNaOHsolution,

ml5.05.0Keepfor10minatroomtemperatureDeterminetheabsorptionat520nm

NormaltubesAbnormaltubesseraSALTsubstratesolutionsera____Waterbathingat37℃for30’Waterbathingat37℃for30’2,4-dinitratephenylhydrazine2,4-dinitratephenylhydrazine____SALTsubstratesolutionWaterbathingat37℃for20’Waterbathingat37℃for20’AddNaOHsolutionandkeepinroomtemperaturefor10’AddNaOHsolutionandkeepinroomtemperaturefor10’Assayat520nmAssayat520nmProfessionalwordsABSTRACTObservingEnzymeActivityChangesofAlanineAminotransferaseinserumObjetcive:TodeterminetheactivityofserumALTinsample.

Method:SALTwasmeasuredbyReitman-Frankelcolorimetricendpointmethod

Results:ThenormalSALTactivityis(??)unit,theabnormalSALTactivityis(??)unit.

Conclusion:TheactivityofSALTinabnormalsampleismuchhigherthanthatinnormalsampleKeyWords:alanineaminotransferase，aspartatetransaminase,enzymeactivity,serum,α-ketoglutarate,substrateAttentions1.吸量应准确，严格控制反应时间和温度。2.在测定SALT活力时，应事先将底物、血清在37℃水浴中恒温3．在测定时，如酶活力较大（大于100