Thelaboratoryexperimentsforthenexttwoweeksdealwiththeenzymeß-galactosidase.Fortheseexperiments,theenzymesourcewillbethebacterium,Escherichiacoli,althoughß-galactosidaseisalsofoundinmanyothermicroorganisms,plantsandanimals,includinghumans.ß-galactosidasecatalyzesthebreakdownofthesubstratelactose(themajorsugarpresentinmilk)totwoproducts,galactoseandglucose,compoundswhichreADIlyfeedintotheglycolyticpathway: 1.Constitutive-thoseenzymesthatarecontinuouslysynthesizedinthecell(e.g.theenzymesofglycolysisorthelacrepressorprotein); 2InducIBLe-thoseenzymesthataresynthesizedinthecellonlywhenaninducer(signal)ispresentinthecell(e.g.ß-galactosidaseortheenzymesinthetryptophanbiosynthesispathway). Thisprocedureisdesignedtoinduceandmeasurethelevelofß-galactosidaseinE.coli.Cellsgrownintheabsenceoflactosedonotsynthesizeß-galactosidase.Ifthesecellsareplacedinamediumcontaininglactose,ß-galactosidaseisproducedwithinminutes,enablingthecellstouselactoseasasourceofcarbonandenergyforgrowth. Somecompoundsareinducersofthesynthesisofß-galactosidaseandsomecompoundsaresubstratesfortheactivityofß-galactosidase.ThisweekyouwillinvestigatetheABIlityoffourcompoundstoinducetheactivityofß-galactosidase.Thefourcompoundsare:lactose,isopropyl-ß-thiogalactoside(IPTG),phenyl-ß-galactoside(PBG)andglucose(GLU). Wewilluseanon-biological(artificial)substratefortheenzyme,Ortho-nitrophenyl-ß-galactoside(ONPG).Inthepresenceofß-galactosidaseONPGisconvertedtogalactoseandOrtho-nitrophenyl(ONP).E.colicellscontainnoenzymescapableofdegradingONPfurther. ONPGiscolorless.ONPisalsocolorlessatneutraloracidpH,butinanalkalinesolutionitisbrightyellow.TheamountofyellowcolorcanbemeasuredinaspectrophotometerandcanbeusedasameasureoftheamountofONPformedinagiventime.SinceONPisaproductofß-galactosidaseactivity,thespectrophotometricmeasurementsmaybeusedasanassayfortheenzyme. a)4mlofstarvedE.colicells(1x107cells/ml). b)0.2mlof.002Minducer(LAC,GLU,IPTG,PBG,ordH2O) Putacaponeachtube,placeina37Cwaterbathandaerate(shake)for30minutes. 1)Onedropsodiumdesoxycholate(1.0mg/ml)2)Onedroptoluene Cap,placeina37oCwaterbathandaerateorshakefor10minutes.Thispreparationmaybeusedforenzymeassays.Keepitinanicebucketandonlyremovesamplewhenneededforassays. 1)2.0mlof0.1Msodiumphosphatebuffer(pH7)2)2.0mllysedE.colipreparation3)0.2mlof0.01MONPG(Substrate)Incubatefor15minutesat37Cwithoutshaking.Stopthereactionbyadding0.5ml2Msodiumcarbonate.Thiswillmakethesolutionalkaline(pH>8)anddenaturetheenzyme.Readtheabsorbanceat420nminaspectrophotometer.Ifthecompoundisaninducer,moreenzymewillbeformed,moresubstrate(ONPG)willbeconvertedtoayellowproductandtheabsorbancewillbehigher.
AlloftheenzymesandotherproteinsinE.colimaybedividedintotwogroupsbasedonwhethertheirproductionisregulatedornot:Enzymeassay:
Asalreadynoted,thenormalBIOLOGicalsubstrateforß-galactosidaseislactose.Intheoryonecanmeasureenzymeactivitybydeterminingtherateofdisappearanceofthesubstrate(lactose)ortherateofformationofaproduct(galactoseorglucose).Inassayingforß-galactosidaseactivity,therearetwodifficultieswitheitherofthesemethods:1)thereisnosimple,rapidandinexpensivewaytoquantifylactose,galactoseorglucose;and2)whenoneisusingacrudecellextractasasourceofß-galactosidase,therearemanyotherenzymespresentthatwillimmediatelyconvertanygalactoseorglucoseformedtoothercompounds.A.GrowthofstarvedE.coli
B.InductionofEnzyme
Thesynthesisofß-galactosidasemaybeinducedusingthefollowingprocedure.Intoalargesize(18mm)labeledtesttubeplace:C.AssayforEnzyme
AlthoughONPGisusedtodeterminewhetherornotß-galactosidasehasbeensynthesizedinthecell,thecompoundwillnotquicklypassthroughalivingcellmembrane.ThereforetheE.colimustfirstbetreatedwithadetergent,sodiumdesoxycholate,andanorganicsolvent,toluene,todestroytheselectivepermeabilityofthecellmembrane.Thistreatment,whichallowsONPGtoenterthecellquickly,alsokillsthecells,butdoesnotaffecttheactivityoftheenzyme.CAUTION!Thesecompoundsarealsotoxictohumans.1.DisruptionofSelectivePermeability:
To4.2mlofaninducedE.colicultureadd:2.EnzymeAssay:
Intoeachsmalllabeledculturetubeplace:
