Lab Techniques & Spectroscopy - Med-Pathway...The Sanger DNA sequencing technique is described and illustrated below. Purified template DNA is enzymatically radiolabeled at the 5’

LabTechniqueswithSpectroscopymed-pathway.comTheMCATexperts


Lab techniques are important for the MCAT, especially as the testexaminesyourabilitytointerpretexperimentaldata.Thesetechniquesare assessed in various parts of themed-pathway diagnostic questionbanks.Thissectionwillexaminehighyieldexperimentalproceduresinboth thebiological andphysical sciences, including spectroscopy. Thefollowingtopicsarecoveredinthiscontentreview:� ElectrophoresiswithReducingandNon-reducingSDSPAGE� Isoelectric Focusing � DNA sequencing � HybridizationTechniques � Generation of libraries � Quantitative PCR andAnalysis of Gene Expression � Site directed Mutagenesis � Western Blotting� Polyclonal&Monoclonal Antibodies� ELISAassay � Chromatin Immunoprecipitation � Flow Cytometry � Chromatography and Protein Purification Tables� Gel Filtration(Size Exclusion) � Ion Exchange � Affinity Chromatography � ThinLayerChromatography(TLC)� GasChromatography� NMRspectroscopy � UV-Visible spectroscopy & Beer Lambert Law � Infrared(IR)Spectroscopy� MassSpectrometryElectrophoresisThebasicanalysisofproteinstructureiscritical forunderstandingthemolecularbasisofhumandisease.Electrophoresisisonesuchanalyticaltool that utilizes an electric field to induce the migration of chargedmacromolecules (e.g. proteins and nucleic acids) through gels(acrylamide for proteins and acrylamide or agarose for DNA) for thepurposesofseparationandanalysis.Thisisshownbelow.Once a voltage drop is established, proteins are accelerated due toelectricforcebutarerapidlybalancedbyagrowingfrictionalforce(fv),resulting inaconstantvelocity. Theresultingelectrophoreticmobility(m) is defined as the velocity normalized by the applied electric field.This property is inversely dependent on the frictional coefficient (f),



whichistheratioofdragforcetovelocityandisapartiallyafunctionoftheobject’ssizeandshape.

Electrophoresis is a common laboratory technique that you willencounter on the MCAT. Proteins are commonly separated inacrylamide gels. The cross-linked acrylamide forms a mesh wheresmallerproteinsorpeptidefragmentsmigratefasterthroughthegelinanelectrophoreticfield.Bothreducingandnativegelsareusedandareshown below for the electrophoresis of a typical antibody(immunoglobulin).Asantibodiesareheterotetramerscomposedoftwoheavyandtwolightchains covalently linked via disulfide bonds (red lines), they havedistinctiveelectrophoresispatternsinreducingandnativegels(Lane1onbothgels:Lane2representssizemarkers).



Treatmentofanantibodywithareducingagentinthepresenceofheatsuch as dithiothreitol (DTT) or β-mercaptoethanol will reduce thecystinebondstofreecysteineresidues(–SH).Thisseparatestheheavyand light chains.When these chains are treatedwith sodium dodecylsulfate (SDS; shown below) and subjected to polyacrylamide gelelectrophoresis(SDSPAGE),theproteinsaredenatured(unfolded)andpossessauniformchargetomassratioandwillthereforemigrateintheelectrophoreticfieldasafunctionoftheirmass.



In the case of a native (non-reducing) gel electrophoresis, proteinsmigrate in their folded state as there is no SDS. In this application,proteinsmigrateintheelectrophoreticfieldasafunctionofthechargetomassratioaswellasafunctionoftheirmolecularshape.Notethatina native gel an intact immunoglobulinmigrates as one species as thedisulfide bonds are still intact. After electrophoresis of proteins inacrylamide gels, the proteins are often visualized by various stainingtechniquesincludingsilverandCoomassieBlue.IsoelectricFocusingIsoelectric focusing isusuallyapplied to separateproteinsbasedupontheir isoelectricpoints, thepHatwhichtheyexhibitanoverallneutralcharge.Thechargeofproteinsconsiderablyvariesasa functionofpH.AthighpHvalues,proteinsoftenexhibitanoverallnegativechargeduetotitrationofcarboxylgroupsandtheneutralizationofbasicgroups:IfpH>pkathentheproteinisnegativelychargedOn the other hand, at low pH values proteins tend to have overallpositive charges as the carboxyl groups have not been completelytitrated and the amine side chains (lysine and arginine as well ashistidine)arestillintheirpositivelychargedform:IfpH<pKathentheproteinispositivelycharged.



In an isoelectric focusing experiment a gel composed of ampholytescreates a stable pH gradient. The gels are createdwith large pores toeliminate separation effects based upon molecular weight. After amixtureofproteins(blackcircle)isaddedtothegel,anelectriccurrentis applied. The proteins will migrate to either the anode or cathodedependingontheirchargeatthepHinwhichtheywereappliedtothegel (usually around neutral pH). In the experiment shown, the redproteinonthelefthasahigherisoelectricpointthanthegreenproteinon the right, suggesting that the red protein hasmore basic residuesthanthegreenprotein.



NucleicAcidTechniques(MolecularBiology)DNAsequencingbytheSangerdideoxytechniqueIn the 1970s,Fred Sangerdeveloped dideoxynucleotide triphosphates(ddNTPs or ddG, ddC, ddA, and ddT) for determining the sequence ofDNA in a sample. These nucleotides have3’ H groupssubstituted forthe3’OH groupnormally used by template-driven DNA polymeraseenzymes (hence, the term"dideoxy"). The 3’OH is an essentialnucleophile for growing nucleic acid chains as shown below (yellowcircles).Normally,the3’OHgroupattackstheα-phosphategroupinthedNTPtobeincorporatedintothegrowingchain.However,whenaDNApolymerase reaction encounters a dideoxynucleotide (ddNTP), thenucleicacidchainisterminateduponincorporationoftheddNTPduetotheabsenceofthefree3’OHgroup.The Sanger DNA sequencing technique is described and illustratedbelow.PurifiedtemplateDNAisenzymaticallyradiolabeledatthe5’end



with the addition of a phosphate group (represented by *). TemplateDNAisincubatedinacocktailcontainingmultiplereactioncomponents.This includes a suitable buffer containing Mg+2and each of the fourdNTPs.Aftermixing in theenzyme(Step1), the final reactionmixture is thenaliquotedintofourseparatetubes(Step2),eachofwhichisspikedwithasingleddNTP. Inasingle tubeeachreactionproceedsuntil theDNApolymerase adds a dideoxynucleotide into the nucleic acid chain.Because both normal deoxynucleotides and a specific dideoxy chainterminating nucleotide are present in a given reaction, a series or“nested set of fragments” is generated for each nucleotide. EachfragmentendswiththeincorporationofachainterminatingddNTP.Aseachfragmentvariesinsize,theycanbeseparatedbyacrylamide/ureagel electrophoresis. In this procedure, small DNA fragments migratefasterthanlargernucleicacidchains.Afterrunningthesequencinggelelectrophoresisexperiment,thefollowingprofileisobtained:



Byreadingthelanesofthegelfromthebottomtotop,theseqeuncecanbedeterminedas:5’-CCGATCTAGG-3’.Therefore,thetemplatesequenceisthecomplementofthis:“5’-CCTAGATCGG-3’.

Nucleicacidhybridization

Nucleic acid hybridization is a fundamental aspect of biology and hasbeen exploited in multiple, medically important techniques. Thisincludes thepolymerase chain reaction(PCR) andSouthern blottingaswell as fluorescent in situ hybridization (FISH). These techniques areoutlinedinthefigureanddiscussedbelow.Hybridization techniquesarecritical foranalyzinggeneexpression. InaNorthern blotting experiment, mRNA molecules are isolated andseparated via gel electrophoresis in a manner analogous to Southernblotting. When single stranded, radiolabeled DNA probes are added,stable, complementary DNA-RNA hybrids can be detected. Therefore,theSouthernblotting technique is fordetectingDNA-DNA interactionsandNorthernblottingisusedtodetectDNA-RNAinteractions.As the Watson-Crick model of DNA shows that the two antiparallelstrands of DNA are held together through hydrogen bonding, DNAstrands are easily separated with heat. The complementary singlestrands of DNA (ssDNA) re-associate after slow cooling. Alternatively,thesinglestrandedDNAmoleculescanbeusedastemplatestoamplifyDNAthroughthetechniqueofPCR(PolymeraseChainReaction).Inthisprocedure(seebelow),primersequencescomplementarytoportionsofthe ssDNAareadded.Themixture is cooled toallowannealing. In thepresenceofDNApolymeraseenzymeandnucleotidetriphosphates,thetemplate DNA is amplified. Afterwards, the amplified DNA is heated,moreprimeranneals,andmoreDNAisamplified.Afterrepeatedcycles,a small sample of DNA can be exponentially amplified to significantlevelsusedinvariousdiagnostictechniques.



Alternatively,thessDNAcanbeprobedfortheexistenceoralterationofvarious target sequences using techniques likeSouthern blotting orFISH. By fixing the single stranded DNA sequences in situ (i.e.membrane or glass slide), radiolabeled or fluorescent probes (i.e.SouthernblotorFISH,respectively)canbeusedtoquerythepresenceofDNAsequences.Suchtechniqueshavewideapplicationsrangingfromforensicsciencetocancerdiagnostics.

WewillexpandfurtheronapplyingthetechniqueofSouthernblottingin theclinical contextofdisease. Imagine that twopatients (AandB)are being screened for the presence of a disease that is caused by adeletioninafragmentofDNA.BelowisaschematicofhowaSouthernblotwouldbesetup in thiscase. The fragmentsofgenomicDNAthataregeneratedafterrestrictiondigestionareresolvedonanagarosegel.This separates DNA fragments based upon their charge-to-mass ratio(i.e. size). Typically, a positive control (+) that uses DNA sequences



known to hybridize via complementarity is used as the probe. Thiscould be from a normal individual. A negative control (-) will nothybridizetothetargetsequenceandthisDNAcouldbederivedfromaspeciesthatdoesnotexpressthegeneorregionofDNAofinterest.Theresolved DNA is transferred to a nitrocellulose membrane and thenprobed with a radiolabeled probe (typically a smaller piece of DNA).TheDNAisdenaturedonthemembrane,allowingfortheprobetobindto itscomplementarysequences. Afterwashing, the filter isdriedandexposedtofilmforautoradiography.Noticethattheprobebindstothesame fragment of DNA in the positive control and in Patient A. Incontrast,theprobebindstoasmallerfragmentofDNA(fastermobilityontheagarosegel)derivedfromPatientB,suggestingthatthispatienthasadeletion.



GenerationofLibrariesThegenerationoflibrariesisspecificallymentionedintheAAMCMCATContentOutline that isavailable for freeonourwebsite.Thisclassicaltechniquewas invaluable for identifying and cloning important genesforbiochemicalanalysis.Although the complete DNA sequencing of the genomes of numerousorganisms has replaced the need for generating certain types oflibraries,thepracticeisstilluseful.Asimpleschematicforgeneratingalibraryisshownbelow.Thegenomeofanorganism(simplifiedwiththeyellowbox)istreatedwith a restriction enzyme (PstI) that recognizes a 6 base pairpalindrome(CTGCAG). Afterdigestionof theDNA,multiple fragmentsofvaryingsizearegeneratedandligatedintoaplasmidvectordigestedwithPstI(showningreens)thatcanreplicateinbacteria.AllofthePstIfragments shouldbe represented in a libraryof plasmids (Onlyone is



showninthepicture).Theligationmixtureistransformedintobacteria.Thosebacteriathathavetakenuptheplasmidareselectedforviadrugresistance (i.e. ampicillin). After isolation of plasmidDNA, restrictiondigestion with PstI is used to confirm the presence of a PstI insertderived from the genome of interest. For this, agarose gelelectrophoresis is used. The DNA can now be studies for variouspurposes.QuantitativePCR&AnalysisofGeneExpressionQuantitative orreal time PCR(qPCR or RT-PCR) is widely used todetermine gene expression levels.Unlike conventional PCR,RT-PCR isquantitative.Theprocedure isshownbelow. InPanelA, totalmRNAisisolatedfromcelllinesthroughtheuseofpolyTaffinitypurification.Asthe vast majority of mRNAmolecules are equipped with poly A tails,poly T affinity purification separates mRNA from other RNA speciesincludingribosomalRNAwhichisabundant.PolyA+RNAiscomposedofmRNAmolecules devoid of introns; only the sequences encodedbythe exons are isolated. The isolated mRNA molecules are used astemplates in reactions catalyzed by reverse transcriptase. Originallydiscovered in retroviruses, reverse transcriptase uses mRNA as asubstratetogeneratecomplementaryDNA(cDNA)molecules.This



reactionoccursthroughanRNA-DNAhybridintermediate.TheenzymeisalsoequippedwithaRNAnucleaseactivitythatdegradestheRNAintheRNA-DNAhybrid,generatingasinglestrandedDNAtemplatethatisused as a substrate for the DNA polymerase function of reversetranscriptase.Panel B shows how the mRNA levels are quantitated. In onemanifestationoftheprocedure,afluorescentprobeishybridizedtotheinternal region of the target gene whose expression levels are beingmeasured.During thepolymerizationstepofPCR, thepolymerasewilldestroy theprobehybridized to the targetDNA thereby liberating thefluorophore from the quenching agent. The amount of releasedfluorophoreisrelatedproportionallytotheamountofinputDNAandismeasuredintheqPCRtechniqueIn qPCR,mRNA is isolated for the generation of complementary DNA(cDNA),whichservesasatemplateformeasuringthelevelsofvarious



genes. In one type of qPCR reaction format, a fluorescent probehybridizes to the target sequence and emits a signal, when separatedfromitsquenchingagent,whichislinearlyrelatedtotheamountofDNApresent. The probe is released from the quenching agent due tonucleotidedegradationviatheactionoftheelongatingDNApolymerase.In eachPCR cycle,DNA is exponentially amplified as a functionof thePCRamplificationefficiency,whichcanvarysignificantlyforeachgeneduetoanumberofexperimentalfactors.SiteDirectedMutagenesisSitedirectedmutagenesisisapowerfultechniqueforgeneratingmutantenzymesandproteinsforstructure-functionstudies.Therearemultiplemethodsavailableforthis.Wewillexamineaversionoftheprocedurethat uses the Dpn I endonuclease, an enzyme that recognizes andcleavesmethylatedDNAsequences.Theschemeisshownbelow.



Aparentalplasmidcontainingthedesiredmutagenesistargetedgeneofinterest is isolated fromabacterialhost that iscapableofmethylatingDNA.Thatis,theparentalplasmidDNAismethylated.Afterheatingandcoolinginthepresenceofmutagenicprimers,PCRamplifactionisusedin the presence of non-methylated nucleotides to generatecomplementarystrandsofDNA.Theprimersmustbecarefullydesignedto intoduce the desired nucleotide substitutions, yet stillmaintain thecapacitytobindtotheplasmidDNA.ThePCR-amplifiedDNAistreatedwithDpnI,aprocedurethatwilldegradethemethylated,parentalDNAthat contains the non-mutated sequence. The remaining plasmid DNAshould be non-methylated. This sample is transformed into a newbacterial host and transformants are screened for the presence of themutation.ANTIBODYTECHNIQUESAntibodies are produced by B cells of lymphoid origin and are keyreagents with a wide variety of clincal applications. In order tounderstand the various procedures that antibodies are used for, it isparamount to understand antibody structure and function. We sawabove the heterodimeric nature of antibodies, but now we need tounderstandthemfurther.The “arms” in the classical Y structure of an antibody possess theantigen binding sites that are encoded in the variable regions. Thevariable region sequences recognize “epitopes” in antigens. Epitopescanbecomprisedofshortsequencesofaminoacidsorcombinationsof



sugars in glycosylated proteins. Therefore, each antibody has twoantigen binding sites. This region is also called the Fab protion of anantibody. Such Fab fragments can be generated through proteolysis.NoticethattheFabprotionofanantibodycontainsbothheavyandlightchainfeatures.The Fc portion of the antibody contains the constant region and iscomposed solely of heavy chain amino acids. Historically, “Fc” means“Frgament crystalliazable” and is often thought of as the tail of theantibody. The constant region of the antibody defines the class ofimmunoglobulin. This is discussed further in the microbiology andimmunologysection.Fornow,wewillexaminetheFcandFabportionofantibodiesinthecontextoftheirutilityinlabatorytechniques.WesternblottingWesternblottingisatechniqueusedtodetectspecificproteinsthroughantibodies that recognize specific epitopes. Epitopes can be unique tospecific proteins or conserved in various proteins. The image belowshows a schematic of a typicalWesternblot that indirectly detects anantigenofinterest.



Step1:Apolyacrylamidegelisloadedwithaproteinsampleofinterestandsubjecttoelectrophoresisinordertoseparatethesampleproteins.In theexample, two lanesare loaded:1)Normal,wild-typesample;2)Mutantsample.Step 2: After electrophoresis the proteins are transferred to amembraneforinsituanalysis.

Step3:Themembrane isblockedwithnon-specificprotein(ie.serumalbumin)toreducenon-specificantibodybindingtothemembrane.Step4:Aprimaryantibody(1°orα)thatrecognizesaspecificproteinis added. The antibody can be either polyclonal (a population ofimmunoglobuins that recognizes multiple epitopes within the same



antigen)ormonoclonal(specificallycreatedtorecognizeoneepitopeinanantigen).Step 5: After incubation to allow for antibody binding, the filter iswashedtogetridofexcessand/ornon-specficallybound1°antibody.Asecondary (2° or α) is added. This antibody specifcally binds to theconstant region of the 1° antibody and is coupled to a fluor (orradiolabel)atitsconstantregion.Step6:Excesssecondaryantibodyiswashedoffandthefilterisdriedandpreparedfordetectionofprotein-immunoglobulincomplexes.NotethatthereisasignalinLane1,butnotLane2asthissampleismissingthe protein due tomutation. Inmany cases, antibodies can recognizetheirepitopesundernon-denaturingconditions.Therefore,SDS-PAGEiscommonly used during elextrophoresis in Western blottingexperiments. However, some antibodies will only recognize theirepitopes under native conditions and the Western blot should bevonductedwithanativegel.ControlexperimentsinWesternBlottingAlthough antibodies can be highly specifc for antigens, non-specificinteractionsarealwaysaconcernandmustbecontrolledfor.Thereareseveralmethodsfordeterminingthis:1) Use of pre-immune serum. Custom-made antibodies are derviedfrom injecting antigen into a host. Use of the serum prior toimmunization should be give no signal, and is therefore considered anegarivecontrol.2)Useofsecondaryantibodyalone.Iftheexperimentisconductedwithomission of the primary antibody, then there should be no signaldetected.Thisisanegativecontrol.



3) Use of recombinant protein. If a known antigen from a proteinextract(i.e.cellline)istobedetectedviaWesternblotting,thenasmallamount of recombinant protein derived from that antigen should beloadedinaseparatelaneofthegelandusedasapositivecontrolforthespecificityandutilityoftheprimaryantibody.PolyclonalandMonoclonalantibodiesPolyclonal antibodies are comprised of multiple immunoglobulinmoleculesthatrecognizeasingleantigenatdifferentsites(epitopes)ofthe molecule. Monoclonal antibodies (MAbs) are generated underlaboratorycircumstancesfromclonalcells(i.e.cellsofthesameorigin)and have a monovalent affinity towards an antigen through therecognition of a single epitope. This is in contrast to polyclonalantibodies that recognizemultiple epitopes on the same antigen. Thegeneration of a homogeneous population ofMAbs represents amajoradvance in medical research and was awarded the Nobel Prize inmedicine.Multiplemonoclonal antibodies have been approved by theFDAforclinicaluse.ThisincludesHerceptin,an

Structure of Her2 receptor. Tyrosine phosphorylation occurs in cytoplasm and is shown in blue.



antibody that targets the Her2 tyrosine kinase and is used for thetreatmentofbreastcancer(Fig.1).MAbs are generated from a cell line (hybridoma) derived from thefusionoftwocelltypes.Thefirstcelltype,aprimaryBcell,cannotgrowon its own, but can produce antibodies. The second cell type, a B cellderivedfromamyeloma,cannotproduceantibodies,butcangrowinitsown.ThestrategyforgeneratingMAbsisasfollows.When transformed, myeloma B cells (or plasmacytomas) deficient inantibody production due to lack of the enzyme hypoxanthine guaninephosphoribosyl transferase (HGPRT) are fused with freshly isolated,primary B cells derived from mice immunized with an antigen ofinterest, hybridoma cell lines are created (see Figure). Sequencescontainingclustersofchargedandpolaraminoacidsareoftenusedasantigens for this procedure, as they are highly immunogenic. TheprimaryBcells,whichcannotreplicateinvitro,produceantibodiesandexpressHGPRT,anenzymeinvolvedinthenucleotidesalvagepathway,a cellular method to generate nucleotides de novo. Fused hybridomacells are cultured in selective HAT media containing the nucleotideprecursorhypoxanthine,thefolateantagonistaminopterin,and



thymidine. Aminopterin inhibits dihydrofolate reductase (DHFR), anenzymeresponsibleforgeneratinguracilfromthymidine.Thispreventsde novo synthesis of DNA, the second pathway for generatingnucleotides. The generation of the hybridoma generates a fused celltype that can synthesize DNA and has unlimited replicative capacity.Moreover, this cell line secretesonlyone antibody type, amonoclonalantibodythatrecognizesoneepitopeonanantigen.



ELISAEnzyme-linkedimmunosorbent(ELISAorELISAassay)isanimportantbiochemicaldiagnostictestusedinlabs.Theyareusedtodeterminethepresenceandamountofvariousproteinsandmetabolites inbiologicaltissues. The direct and sandwich ELISA will be discussed here. Aschematicisshownbelow.InthedirectELISA,asolutioncontainingtheantigentobemeasuredisplaced in a plastic coated well (solid state support) (STEP A). Theantigen adheres to the surface through charged interactions with thewell.Afterwards,theplateiscoatedwithaprotein(i.e.serumalbumin)to saturate all binding sites. After washing to release all unboundmaterial,aprimaryantibodyisaddedthatisspecificfortheantigenofinterest (STEP B). After washing away the unbound antibody, asecondary antibody is added (STEP C). Importantly, the secondaryantibody recognizes the constant region of the primary antibody (not



thevariableregion)andislabeledwithaflour(*)thatcanbedetectedand quantitated. In some cases, the primary antibody can be labeledwith a flour. In some cases, the secondary antibody is linked to anenzyme thatperformsa reaction that generates aproduct that canbedetectedbyaspectrophotometer.Hence, thename“enzyme-linked” inELISA.Wewillusethename“flour”asageneralterm.The sandwich ELISA uses two antibodies that recognize differentepitopes of the same antigen. In the sandwich ELISA, a “capture”antibody specific foranantigenof interest is added to thewell (STEPA).Afterbinding,serumalbuminisaddedtoblockallpotentialbindingsiteson thesurface.Afterwards, thewell iswashedandamixture(i.e.cellextractorserumsample)containingtheantigenofinterestisaddedto the well. This allows for the formation of an antibody-antigencomplex (STEP B). After washing, a second antibody specific for theantigen is added to thewell. This antibody is labeledwith a fluor fordetection.Thebindingofbothantibodiestotheantigenformsthebasisofthe“sandwich”(STEPC).



ChromatinImmunoprecipitation(ChIP)In addition to using antibodies as reagents in Western blottingexperiments, researchers also use them for immunoprecipitation (IP)experiments.Chromatin immunoprecipitation (ChIP) assays are used to determinethelocationofDNAbindingproteinsalongthelengthofthechromatinfiber. They can also be used to determine the nature of histonemodifications at a particular DNA locus. This includes examining thestateoflysinemethylation,includingtrimethylationatlysineresidue9(K9me3). The experiment shown above used an antibody thatspecifically recognizes lysine 9 when it is tri-methylated. ChIP assaysinvolve: 1) the treatment of cellswith formaldehyde to allowprotein-DNA crosslinking; 2) sonication of the cells to fragment the DNA; 3)immunoprecipitationoftheprotein-DNAfragmentsusingantibodies,4)

K9 K9me3 K9 K9

-formaldehyde crosslink

-sonicate

nucleosomes

K9

K9me3

K9

K9

Antibody to histone H3 K9me3

K9me3

Immunoprecipitation -Digest Protein -Purify DNA

-Perform quantitative PCR with gene-specific

primers



PCRusingspecificprimerstoquantifytheassociatedDNA.TheresultsofChIPanalysisofhealthyversusFSHDpatientcellsareshownabove.ThepresenceofPCR-amplifiedDNAsuggeststhatthisDNAisassociatedwithhostnesthatcontainthetri-methylationmarkat lysineresidue9.Non-specificIgGantibodies,aswellastheadditionofnoantibody,serveasnegativecontrols.FlowcytometryFlow cytometry is used to detect specific characteristics of cells insolution.Itisoftenusedtodetectthepresenceofcertaincelltypesinabiological sample. For example, imagine if a doctor was trying todiagnose a patient with mastocytosis, a condition where mast cellsinappropriatelyaccumulate in thebonemarrow.AsmastcellsexpressCD117ontheirsurface,treatmentofthesamplefromthebonemarrowwithananti-CD117antibodywouldlabelmastcellswiththeantibody.Ifthe antibody is fluorescently labeled, then the flow cytometer canspecificallydetectandquantitatetheCD117+cellsinapopulation.Flow



cytometryisalsousedtomeasurecellsizeandpurifiedcellpopulations.In the procedure, a cell suspension is labeled with a fluorescentantibody and applied to the flow cytometer. The cells funnel down achamberinsinglefileandpassthroughalaserbeamoflight.Asthelightshines on the passing cells, those cells possessing the specificfluorescent label will emit light that can be used to quantitate thenumber of cells in the population. In addition, light is scattered in aforwardandside fashionandthis information isalsousedtogenerateinformationregardingcellsize.

An example of a flow cytometry experiment is shown above. In thisexperiment,cellsweretreatedwithionizingradiation(IR).Asacontrol,a setof cellswas setasidebefore treatment. After72hours, the cellswere prepared for flow cytometry through treatmentwith propidiumiodide (PI), a fluorescentdye thatbinds to and identifiesDNA, andbyadditionofanantibodyagainstAnnexinV,amarkerforapoptosis.



Eachpixelintheflowcytometryexperimentshowsacellthatisstainedwith either PI and/or Annexin V. The experimental data show twothings:1)theamountofDNAasmeasuredbythePIsignalremainsthesame after treatment and; 2) the amount of Annexin V stainingincreases after treatment. Therefore, one can conclude that thetreatment of the cells with IR induces apoptosis as witnessed by theincrease inAnnexinVstaining.However, the totalamountofDNAhasremainedthesameduringthe72hours.Chromatography

Chromatography is a commonlyused technique for thepurification ofproteinsandpeptides. This techniquehasmanyapplicationsbuteachone utilizes a stationary phase (i.e. beads on a column, silica plate)capable of interacting with a solution of molecules (cell extract).Depending on the nature of the stationary phase (charge, polarity,hydrophobicity), molecules will either interact and bind, or passthrough with the mobile phase. This is the basis of separation: theintrinsic differences in physical properties. We will examine variousformsofchromatography,startingwithproteinpurification.

ProteinPurification

Proteins and peptides are purified by various means that exploitdifferential physical properties, including size, charge, and polarity.Proteinpurificationtablesareoftengeneratedinordertoevaluatetheefficiency of various steps in the purification process. A hypotheticalpurificationtableforanenzymeisshownbelow.

1. Each major purification step is described and the total amount ofproteinisdeterminedbyasuitableassay(i.e.Bradfordassay)islisted.The vastmajority of purification schemes beginwith a crude, solublelysate derived from a cellular source. Crude extracts are commonlyderived from the supernatant after a sample is centrifuged. In somecases,proteinsmaybeassociatedwiththemembrane(i.e.peripheralor



integral). In these cases, the membrane fraction (pellet) is eitherwashedorsolubilizedandthenactivityismeasured.

2.Determinationoftargetactivity.Inthetableshown,enzymeactivity,asdeterminedviaanenzymeassay, is listed inunits thatare typicallyidentified by the amount of activity (i.e. cleavage of DNA for anendonuclease) per unit time. The temperature, pH, and bufferconditionsarealsotypicallyincluded.

Insomeproteinpurificationschemes,anenzymeisnotthetargetofthepurification. In these cases, the activity is often more difficult todetermine.Intheeventthatnoquantitativeassayisavailable,onewayto determine activity is through either silver or Coomassie staining ofthe gels. After scanning, a quantitated value is determined for theproteinbandofinterest(ifitisknown).

In the case of purification schemes for targets that have quantitativeassays, specific activity is always included in the table. The specific



activity is derived from the ratio of the activity (enzyme units)/mgprotein.

4.Thepercentyieldforeachstepiscalculatedbydividingtheyieldforagivenstepbytheyieldinthefirststep(alwaysgivenas100%).Thefoldpurificationforeachstepisdeterminedbydividingthespecificactivityofeachstepbythespecificactivityforthefirststep.

Three forms of chromatography exploiting these properties aredescribed below. For the two examples of column chromatography,both high and low (i.e. gravity) pressure can be applied to move asamplethroughanabsorptionmatrix.

Gelfiltration(Sizeexclusionchromatography)

Agarosebeadswithdefinedpore sizesareused to separatemoleculesbased upon differences in their molecular weights. This technique ismostaccuratewithglobularproteins.Astheproteinsflowthroughthecolumn,lowermolecularweightproteinsare“trapped”withintheporesof the agarose matrix. They are said to be “included”. However, the



higher molecular weight forms cannot fit inside the pore. They aretherefore considered “excluded”. In gel filtration, larger proteins elutefirstandthesmallermolecularweightproteinselutelater.

IonexchangechromatographyIon exchange columns are composed of a solid matrix coated with acharged species. As amino acids, peptides and proteins havemultiplecharges(polyacids),theywillexhibitanoverallchargeatanygivenpH.Apositivelychargedmatrixexchangesanionsandanegativelychargedmatrixexchangescations.

For this example, two species with differing, yet overall negative,charges are loaded onto a positively charged matrix. This anionexchange column will bind the negatively charged species. Any netpositivelychargedspeciespresentinthesamplewouldnotbeexpectedtobind.Itwouldthereforebefoundintheflowthroughfractions.Afterbinding, thecolumn isexposed to increasing levelsof salt (NaCl).Thisagentservestodisrupttheinteractionbetweenthenegativelychargedprotein species and the column matrix. As the two column-boundspecies vary in the magnitude of their negative charge, the leastnegatively charged species would be expected to elute first from thecolumn.Thisisindependentofthesizeofthemolecule.

Takethecaseofasolutionofthreeaminoacids(ASP,HIS,andLYS)atpH = 6.0 that was subjected to a cation exchange column (negativelychargedmatrix).Afterbindingoftheaminoacids,apHgradientwasuseforelution?Whatistheorderofelutionofeachaminoacid?



Appreciate that a cation exchange column is composed of a matrixconsistingofnegativelychargedspecies(seeimagebelow).ThismeansthatthosemoleculeswiththeleastpositivechargeatpH=6.0(thepHofthe buffer) will elute first. In other words, the least basic species (ormostacidic)willelutefirst.AtpH=6.0,aspartatehasanetchargeof-1andsincethepKaofthehistidinesidechainisapproximately6.0,then1⁄2 of the histidinemoleculeswill be charged and the remaining 1/2will be uncharged (determined from using the Henderson Hasselbachequation).Therefore, thenet chargeofhistidineatpH=6.0 is~+0.5.BothnitrogengroupsinargininewouldbepositivelychargedasthepKafor each group is approximately 12. The net charge on arginine istherefore+1 (-1+1+1=+1).Therefore, the amino acids in orderofincreasingpositivecharge(basicity)areASP,HIS,ARG.

ThebindingstepoccursatpH=6.00.AtthispHthenegativelychargedASPwouldbindtothecolumnwiththeleaststrengthasithasonlyonepositively charged nitrogen and two negatively charged carboxyl



groups,ASPelutesfirstfollowedbyHISandthenARG.

AffinitychromatographyBesides gel filtration and ion exchange, affinity chromatography is amajor method for protein purification. One common use of thistechnique is to affinity purify antibodies. Let’s take the case where aresearchergeneratedanantibodyagainstapeptidesequenceinatargetproteinofchoice.Inthefirststep,thechemicallysynthesizedpeptideisinjected into a host. This will elicit an immune response and thegeneration of antibodies against the peptide. The antibody (red) iscollectedfromtheserum,buttherearenumerousadditionalantibodiesintheserumrepresentedbygreen,blue,andpurplestructures.Affinitychromatographyisusedtoseparatetheantibodyofinterest(red)fromtheotherantibodies.Forthis,thepeptideantigeniscovalentlylinkedtoagarose beads by any of several techniques not discussed here. Aftergeneration of this affinitymatrix, the serum is applied to the column.After binding, the column is washed to dislodge any non-specificinteractions. Only those specieswith affinity towards the peptidewillbind to the column; the other antibodies will flow through. Theantibodiesspecific for thepeptideof interestareelutedbyadditionofexcesscompetitorpeptidetothecolumn.Thisisthesamepeptidethatwasusedtogeneratetheantibodies.TheantibodiesarecollectedintheeluateandassayedbyWesternblotting.



LivingcellsarefirsttreatedwithformaldehydetocrosslinkproteinstoDNA. This“freezes”whateverproteinsareassociatedwithDNAatthetime of chemical addition. The cells are then lysed and subject tosonication with high-energy sound waves that shear the DNA. Animportantconsiderationofthisstepistogeneratearelativelyuniformdistribution of protein/DNA fragments so that regions of DNA can beisolated (thus, choice A is incorrect) that is of equal relativeconcentration between the two states that will be compared (makingchoice B incorrect). A specific antibody is then added to theDNA/protein fragments (choice C is incorrect) and is then used forimmunoprecipitation (IP). Following the IP step,proteins aredigestedbeforeDNApurification.TheDNAisthensubjectedtoPCRusinggene-specificprimers. The lengthof the twoprimersused in thePCRdoesnot have to be identical, only that they are specific (choice D iscorrect).



PROTEINPURIFICATIONSAMPLEPASSAGEAlysosomalenzyme,tentativelyidentifiedasX123,wasoverexpressedin a mammalian cell line in preparation for purification. The enzymewasbelievedtobestableandtohydrolyzeproteinsthroughrecognitionofaKFERQsequencemotif.Theexperimentalpurificationschemewasdescribedasfollows.As shown in the schematic,2.0LofHeLa cellsweregrown in spinnerflasks, harvested, and pelleted by centrifugation. The cells wereresuspendedinphosphatebufferedsaline(PBS;0.150MNaCl,3.0mMKCl, 1mM Na2HPO4, 2.0 mM KH2PO4, pH = 7.4) and sonicated, aprocedure that disrupts cell membranes and membrane-boundorganelles.Thecrudeextractwasobtainedandcentrifugedat12,000gfor10minutesat4°C.Thesupernatantwasappliedtoacationexchangecolumn. After binding, the column was eluted with Tris-based buffergradient ranging from pH 7.4-10.0. 0.3 mL eluates were assayed forproteincontentbymeasuringopticaldensityat280nm.Fractions10-15 were pooled, dialyzed in hydrolase buffer, and applied to a gelfiltration column. A single peak was observed from this column (notshown).Enzymeactivitywasonlydetectedforfractions10-15.Thedatawereplottedinapurificationtable.



Asecondpurificationschemewasdesignedtoimprovetheyieldoftheenzyme. In this experiment, a new step prior to the anion exchangecolumnwasintroduced.Forthis,thesaltconcentrationofthecelllysatewasadjustedto1.0MwithKClandthisnewsamplewasdirectlyappliedtoahydrophobiccolumn.Afterelutionofboundprotein,fractionsweredialyzed in enzyme buffer. Active fractionswere pooled and both the



cation exchange and gel filtration steps were performed as describedabove.Thepurificationtableisshownbelow.

PASSAGEQUESTIONS1.Whichofthefollowingenzymeactivityvs.pHprofilesbestdescribesthepurifiedtargetenzyme?

A.ProfileIB.ProfileIIC.ProfileIIID.ProfileIV



2.Duringthesecondpurificationprocedure,ahydrophobiccolumnwasused.Boundproteinismosteffectivelyreleasedthroughwhichofthefollowingprocedures?A.Decreasingsaltgradientfrom1.0Mto0.1MNaClB.IncreasingpHgradientfrom4-10.C.Increasingsaltgradientfrom0.1Mto1.0MNaClD.DecreasingpHgradientfrom10-4.3.TheincorporationofthehydrophobiccolumnimprovedtheenzymeX123purificationscheme.Whichofthefollowingstatementsbestdescribesthereasonsforthis?A.Decreasedbindingoflysosomalenzymestocolumn.B.RemovalofinhibitoroflysosomalenzymeX123.C.Emulateshydrophobicenvironmentoflysosome.D.Bindshydrophobicresiduestostimulateenzymeactivity.4.Thefoldpurificationafterthecationexchangecolumninthefirstpurificationschemeisapproximately:A.2.0B.3.0C.6.0D.12.0



ANNOTATIONSToProteinPurificationPassage1.ChoiceAiscorrect(ProfileI).Althoughthepassageneverexplicitlystates the precise enzyme assay conditions, appreciate that lysosomalenzymeshaveoptimalpHactivityaround4-5.OnlyprofileIconformstothis.ChoiceBiswrongbecausethepHoptimumis~8.0,wellabovetheoptimal pH for the acidic lysosome. Do not confuse the fact that theenzymeelutedfromtheionexchangecolumnaroundpH9.0(fractions10-15).ThisreasoningcanalsobeusedtoeliminatechoiceC,aprofilethathas themostenzymeactivityat thepH~9.0. ChoiceD iswrongbecausenoenzymeactivityisdetectedbetweenpH4-5.2. Choice A is correct. Hydrophobic chromatography exploits thehydrophobiceffect.Wewilldescribethisbelow,butyoucouldalsoinferwhat the answer choicewaswithout any extended knowledge of thisphenomenon.First,notefromthepassagethatthesaltconcentrationofthelysatewasincreasedto1.0MwithKCl.Thissuggeststhathighsaltconcentrations favor the binding of proteins to hydrophobic columns.Therefore,byimplication,reducingthesaltconcentrationwillpromoteelutionofproteinfromthecolumn.The hydrophobic effect describes the orientation of water moleculesaround a hydrophobe. In the case of hydrophobic chromatography,proteinsbindtothematrixinareversiblefashionasafunctionoftheirhydrophobicity. The column contains solid state, hydrophobicstructuressuchasphenylsepharose(yellowhydrophobe).Inalowsaltenvironment, water molecules orient themselves around thehydrophobeinanorderlyfashion.(Wateralsoorientsitselfaroundtheproteinsolutestoo,butthisisnowshown).However,inthepresenceofsalt fromthebuffer, thesolvationof thehydrophobe isreducedas thewater can now interact with the salt ions. As the solvation of thehydrophobe decreases, hydrophobic regions of the protein becomeexposed,causinghydrophobicinteractionsbetweenthoseproteinswithexposedhydrophobicsequencesandthestationaryhydrophobethatis



partofthecolumnmatrix.Asaresult,thereismoredisorganizationofwaterstructure,demonstratingthatthehydrophobiceffectisdrivenbythermodynamics(i.e.entropy).

3.ChoiceB is thecreditedanswer.Asthepurificationofalysosomalenzyme was conducted from cell extracts, as opposed to purifiedlysosomes,onemightexpectthepresenceoffactorsthatcouldbindandto X123 and interfere with its activity. In principle, the hydrophobiccolumn could purify such factors away from X123. Further, thepurification of X123 from whole cell extracts could also explain theapparently low specific activities observed in the purification table.ChoiceAisincorrectbecausethecolumnwouldbeexpectedtoincreasethe binding of lysosomal enzymes. Choice C is incorrect because thelysosomehasanacidicenvironment,notahydrophobicone.ChoiceDisincorrectbecausetheenzymeassay(andthereforeitsdetectedactivity)isperformedinsolution,notonthecolumn.4.ChoiceBiscorrect.Thefoldpurificationforeachstepisdeterminedbydividing thespecificactivityofeachstepby thespecificactivity for



the first step. As calculated from the table, the fold purification is:12.15/4.19=2.89.

ThinLayerChromatography)

TLCisusefulforseparatingaminoacidsandsmallpeptidesbasedupondifferencesinpolarity.Detectionisdeterminedthroughradiolabelingorstaining techniques suchasninhydrin for aminoacids. In theexampleshown, a sample containing two molecules that vary in polarity arespottedintoacellulosecoatedTLCplate.Theoriginismarkedasshown.Afterwards,thesamplesareimmersedintoatroughofbutanol(amorenonpolarmedium).Throughcapillaryaction,themoleculesmoveuptheplate. However, as polar molecules interact more favorably with thepolar, cellulose surface, their mobility is hindered relative to morenonpolarmolecules that unfavorably interact with thematrix. This ishowseparationisachieved.Bydeterminingthedistancetraveledfromthe origin as a function of time, the ratio to front (RF) value can bedetermined.ArelativelylowerRFvalueindicatesamorepolarmolecule.

AsTLC separates thingsbasedupon theirpolarity and themostpolarmoleculeswillbeclosest to theorigin.This isbecause “polarmigrateslowerandslower”.The silica surfaceof theTLCplate ispolarand the



polar molecules will interact more with the surface compared to arelativelylesspolarmolecule,retardingtheirmigrationuptheplate.

GaschromatographyRecallthatlikemanyformsofchromatography,therearetwophases:amobile phase and a solid, stationary phase. In gas-liquidchromatography, the mobile phase is usually an inert gas and thestationaryphaseisaliquid-gelphase(ahydrophobicresininthiscase)thatcoatsthewallsofthecolumn.Inagaschromatographyexperiment,asampleisinjectedtothecolumnwhere it is immediatelyvaporizedwithheat.Asgas is introduced intothecolumn,thevaporizedmoleculesarecarriedintothecolumnwheretheycaninteractwitheitherthegaseousorliquidphase.Theamountoftimeamoleculeinteractswiththestationaryphase(liquidgel)dependsonitsvolatility.Theretentiontimereflectsthis;higherretentiontimessignifyastrongerinteractionwiththeliquidphase.Thiswouldtranslateinto having a higher boiling point (less volatile). In this separationtechnique,themostvolatilecomponentwillbedetectedfirst.



Spectroscopy

Spectroscopy is a powerful method for determining the structure ofmolecules. Most molecules exist in the grounded state, their lowestenergeticform.Additionofenergytothemolecule, intheformof light(i.e. photon) and/or energy, often causes some of the bonds (i.e.functionalgroups)within themolecule toabsorbenergyandadoptanexcitedstate.Atsomepoint,themoleculewillreturntothegroundstateand release light or energy. This section examinesmultiple forms ofspectroscopy that will appear on the MCAT. This includes nuclearmagnetic resonance (NMR), infrared (IR)spectroscopy,andultraviolet(UV)spectroscopy.

NMR

NMRwasdevelopedbyscientists inorder to studyatomicnuclei.Onemajor application of NMR is to determine the structure of molecules.Nuclei spinandgeneratemagnetic fields. In theabsenceof anappliedmagnetic field, the nuclei are randomly oriented. However, in thepresenceofamagneticfield,thenucleialigneitherwithoragainstthefield (spin up or α and spin down or β). The α and β spin states areseparatedbyanamountofenergythatisproportionaltothestrengthoftheappliedmagneticfield(BO).

Stationary phase Gas

Injector Gas flow Control

Detector

Recorder Column



NMR spectra can be generated for various types of atomic nuclei, buttheMCATwillexaminefourmajorpropertiesassociatedwith1HNMR:1)ChemicalEquivalencyofProtons2)TheChemicalShift3)Splitting4)Integration1)NMRSpectrumSignalsandChemicalEquivalencyofProtonsProtons(orhydrogenatomsinthevernacularofNMR)thatexistinthesamemolecular environment are referred to as chemically equivalentprotons.Benzene (C6H6) isone suchexampleand its spectra is shownbelow.Becauseallof thehydrogenatomsofbenzeneare inequivalentenvironmentsduetothesymmetryofthemolecule,theyeachgeneratesignalsof the same frequency.Therefore, there is onlyonepeak in itsNMRspectra.



Incontrasttobenzene,thehydrogenatomsin1,4dichlorobutaneexistin twodistinct environments as shown.Becauseof this, there are twodistinctNMRpeaksfor1,4dichloroethaneasshownbelow.



2)TheChemicalShiftThepositionofthetworesonancepeaksfor1,4dichlorobutaneiscalledits chemical shift value. The phenomenon of “shielding” underlies theprincipleofthechemicalshift.RecallthattheunderlyingconceptbehindNMR is themagnetic behavior of the nucleus of the atom (hydrogen).However, as the nucleus of an atom lies behind a cloud of swirlingelectronstheappliedmagneticforceisreducedfromthisphenomenon.Thoseatomsthatcontainmoreelectrondensity in theenvironmentoftheproton(hydrogenatom)beingexaminedviaNMRspectroscopyaremore shielded from the applied magnetic force. Those protons thatexperience a smaller appliedmagnetic fielddue to shielding require alower resonance frequency. They are said to be “upfield” in thespectrum. This explainswhy the α protons in 1, 4 dichlorobutane aremore“deshielded”andhaveamoredownfieldshiftthantheβprotons;theelectronegativechlorinegroupspulltheelectrondensityawayfromtheprotonsanddeshieldit.Allchemicalshiftsaremeasuredrelativetothestandardtrimethylsaline(TMS).



Additional factors influence the chemical shift. For example, themoreacidicahydrogenatomis,themoredeshieldeditis.ThisisparticularlytruewhenthehydrogenisbondedtoanelectronegativeatomsuchasOorN.Theelectronegativityoftheseatomspullstheelectronsawayfromthe hydrogen atom, creating a deshielding effect that lowers theresonance frequency. Further, as hybridization influences acidity, thehydrogenatomsinethylene(CH2=CH2)aremoredeshielded(relativelydownfield)relativethoseinethane(CH3CH3).A tableof relative chemical shift values for some important functionalgroups are shown. The values are approximations and expressed inpartspermillion(ppm).Notethatthemoreacidicahydrogenatomis,themoredownfielditsresonancefrequencyis.Thisincludestheacidichydrogen atoms that are stabilized by resonance (aldehydes andcarboxylicacids).



3)SplittingSpin-spin splitting patterns are observed when hydrogen atomsindifferentelectronic environments interact with each other. This ismost prominentwith hydrogen atoms on adjacent atoms. The rule torememberisthen+1splittingrule:thelevelofsplittingisafunctionofthenumber ofnonequivalent, neighboringhydrogen atoms, or n.Notefrom the structure of 1,4 dichloroethane that both the alpha and betahydrogenatomsareeachadjacent to2nonequivalenthydrogenatoms(n=1),meaningthateachpeakwillsplit intoa2+1=3,or triplet.Acloser examination of the spectra presented above for this moleculeshowsthatitisindeedatriplet.4)IntegrationIntegration is the measurement of the area under the peak and isproportionaltothenumberofprotonsthatgaverisetothepeak.NMRProblemBelowistheNMRspectrumofchlorocyclobutane.Assignthehydrogenatomsatpositions1-4withpeaksA,B,andC.



Immediately appreciate that the hydrogen atoms at positions 2 and 3are in identical chemical environments due to symmetry. There are atotal of four hydrogen atoms that contribute to this peak. This iscontrast to the hydrogen atoms at positions 1 and 4 that give rise topeaks composed of two hydrogen atoms. Therefore, through theprincipleofintegrationpeakBiscomposedofthefourprotonsbondedto carbon atoms 2 and 3. Next, observe that the hydrogen atoms atposition1and4differthroughthepresenceoftheC-Clbondatposition1.Becauseof the strongpolarbond, thosehydrogenatomsbonded tocarbon 1 will be deshielded and have a downfield shift and willrepresentpeakC. Thehydrogenatomsbondedtocarbon4willbethemostshieldedand lies furthestupfield inpeakA.Note thateachpeak,thoughtheprincipleofsplitting,appearsintheformofamultiplet.Ultraviolet(UV)andVisiblespectroscopyUV(180-400nm)andvisiblelightpossesstherightamountofenergytopromote an electron from one molecular orbital to another. UVspectroscopy reveals structural information about molecules,particularlytransitionstatemetalsandthosecompoundswithπbonds.Thetechniquecandetectbondingandnonbondingelectrons.Molecules that absorb light with the energy necessary to excite anelectron to a higher molecular orbital enter the “excited state”. Fortransitionmetalsionsinsolution,thedorbitalsabsorbvisiblelightandchange colors. The absorbance spectrum for acetone is shown below.Acetone contains a carbonyl chromophore: the region of themoleculethatabsorbsUV/visiblelight.Asacetonecontainsbothπelectronsandlonepairelectrons, therearetwoabsorbancepeaks:Amajoronenear190nmandasmalleronerepresentingthelonepairelectronsnear270nm.Themajorpeakisreferredtoasthe“lambdamax”orλmax.



TrendsinUV/VisibleSpectroscopy:EffectsonλmaxThe absorption spectra of molecules change as a function of theintroduction of more conjugated double bonds and the availability oflonepairsofelectronstointeractwithπbonds. Theeffectofthisistoreduce the energy required to achieve the transition from the groundstate to the excited state. As energy is inversely related to thewavelength,theλmaxincreases.Theimagebelowshowsafewtrendsthatyoushouldbefamiliarwith.InRowA,theintroductionofconjugateddoublebondsfromethyleneto1,3butadieneincreasestheλmax.InRowB,theionizationofphenoltothephenolateiongeneratesalonepairthatinteractswiththeconjugatedπelectrons in the parental benzene ring. In RowC, the loss of a protonfromanilineathighpHgeneratesanaminewithalonepairofelectronsthatcaninteractwiththeconjugatedπelectronsinthering.



ApplicationofUV/VisibleSpectroscopyAstheabsorptionspectraofamoleculecanchangethroughthelossofprotons, this property canbe exploited in enzymeassays such as thatforlactatedehydrogenase.Pyruvate+NADH+H+! Lactate+NAD+

The λmax of NADH is 340 nm. Therefore, through monitoring thedecrease in absorption at 340 nm, the reaction catalyzed by lactatedehydrogenasecanbemonitored.TheBeerLambertLawThe amount of electromagnetic radiation absorbed and emitted bycompounds canbeused toanalyze the structuralnatureofmolecules.



This is because the absorbance of a compound depends on theconcentrationofabsorbingspecies(chromophores)insolution.Whenmonochromatic radiation of initial intensity (IO, photons s-1) isdirected at a solution (protein or nucleic acid) containing solutemolecules, light is absorbed and emitted with an intensity of I (Seebelow).The%oflightinthebeamthatreachesthedetectoriscalledthe%transmittance(%T).However,mostmeasurementsuseabsorbance:(A) = -log IO/I. The absorbanceofmolecules is dependent onmultiplefactorsandisdescribedbytheBeer-LambertLaw:A=εclε=themolarextinctioncoefficient(litermol-1cm-1);c=concentration(mol/litre);l=pathlength(cm).



Infrared(IR)SpectroscopyInfrared (IR) radiation is used to examine molecules through theidentification of their functional groups. IR radiation has theappropriate amount of energy that corresponds to the stretching andbending frequencies that are characteristic of various covalent bondswithinmolecules. The frequencyof electromagnetic radiationused inIRexperiments isexpressed in thewavenumber (ν),which representsthenumberofwavespercm.Theunitofthewavenumberiscm-1.Theinfraredspectrumrangesfrom4000-6000cm-1.Befamiliarwiththecommonfunctionalgroupsandtheircorrespondingstretchingfrequencies.Thetablebelowdescribesthisfurther.



The IR spectra for ethanol is shown below; note the presence of thebroadalcoholpeak.

NotableTrendsinIRSpectraTherearesomenotabletrendsinIRspectrathatareworthmentioning:1) As the dipole moment of the bond increases, the intensity of theabsorptionincreases.2)Lighteratomsshowabsorptionbandsatlargerwavenumbers.3)Thestrongerthebond,themoreenergyisrequiredtostretchit.Thisisbecauseheavieratomsvibrateatlowerfrequencies:C=C(1650cm-1)>C-C(1200cm-1)



MassSpectrometryTheMCATContentOutlinespecificallylistsmassspectrometryor“massspec”. This powerful technique is used in research and clinical labs toidentifythepresenceofcompoundssuchaspeptides.Initially,asamplesuchasamixtureofproteinsisdigestedbyproteases,creating a complex mixture of peptides that are separated by liquidchromatography (LC). Thesesamplesare thenvaporizedand injectedinto the MS device a shown below. In this first step, or ionization,removalofoneormoreelectronsfromatomsgeneratesapositiveioniccharge within the atoms that make up the peptide fragments. Theionizationstepestablishestheoverallmasstochargeratio(m/z)ofthepeptides.Thepositivelychargedpeptidefragmentsarethenacceleratedso that theyhave the samekineticenergy. Thenext step involves theuse of an electromagnet and results in deflection of the peptides as afunctionof theirmassand theiroverallpositivecharge. An importantfeatureofthisstepisthedegreeofdeflectiondependsonthem/zratiobut the electromagnet itself does not determine the m/z ratio. The

Vaporized Sample

Ionization

Acceleration

Deflection

Electromagnet

Detection



deflectedpeptidesaredeflectedanddetected,allowing themass tobecalculated based upon the amount of current produced. A massspectrum is shown that plots the relative abundance of the detectedpeptide vs. the charge to mass ratio. Mass spec is a highly sensitivetechniqueandcandistinguishbetweenisotopesofthesameatom.

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Lab Techniques & Spectroscopy - Med-Pathway...The Sanger DNA sequencing technique is described and illustrated below. Purified template DNA is enzymatically radiolabeled at the 5’