1. Lysozyme was purified from chicken egg whites using cation exchange chromatography. Fractions were collected as salt concentration increased and tested for lysozyme activity. 2. Bradford assays determined protein concentrations of samples, allowing calculation of lysozyme extract concentration and specific activity. 3. SDS-PAGE analysis showed lysozyme was present and purified in fractions 10-13 based on comparison to protein standards. While some lysozyme appeared in egg extract and flow-through, fractions had greater concentration and purity.

1. Lorraine Salterelli
BiochemistryI
11/23/2015
Working with Lysozyme
Results/Calculations
A. CM-52 Column Results
Figure 1. Absorbance of Fractions In Lysozyme Purification representsthe absorbance observed
(primaryyaxis;on the left) forvariousfractionsof flow-through(blue diamonds)andthe
purifiedlysozymerun-off(redsquares).The flow-throughwasobtainedusingaminimal salt
concentrationof 0.025M NaCl,as shownbythe greentriangles.WhenSaltconcentration
increased(secondaryy-axis;onthe right) the lysozyme purificationbegan. Volumes from1mL-
0
0.2
0.4
0.6
0.8
1
1.2
0
0.5
1
1.5
2
2.5
3
3.5
0 5 10 15 20 25 30 35
[NaCl](M)
Absorbance(280nm)
Volume (mL)
Absorbanceof Various Fractions Observed during
Lysozyme Purification
Flow through
Purified Lysozyme
Increasing Salt

2. 10mL representedflow-through,whereasvolumes11mL-30mL representedpurifiedlysozyme
activity.Anassaywas performedonvariouspurifiedlysozyme fractionstotestforpresence of
the enzyme.The fractionsthatfall withinthe redbox (identifiedasfractions 9-10) hadthe
greatestlysozyme activity.
B. ProteinConcentrations(Bradford Assay)
Table 1. Concentration ofSamples Derivedby Bradford Assay showsthe average absorbance
observedforeachsample,the calculatedmass(foundusingthe line of the equationderived
fromlinearregressionof the absorbance of standards),the volume of the sample addedtothe
assay,and the concentrationof samples.
Sample Avg Abs (nm) Mass (µg)
St.
Dev
Vol. Added
(µL)
Concentration
(µg/µL)
Extract (undilited) Too Conc for Assay, see sample calculations. 134.2*†
Extract (diluted) 0.332 3.36* 0.257 5.0 0.671*
Extract/4 0.268 2.67 0.272 2.5 1.07
Flow-thru (undiluted) Too Conc for Assay, see sample calculations. 48.2
Flow-thru (diluted) 0.345 3.50 NA 10 0.350
Flow-thru/2 0.131 1.204 0.053 5.0 0.241
Fract. 10 0.073 0.584 0.107 25 0.0233
Fract. 11 0.064 0.487 0.130 25 0.0195
Fract. 12 0.061 0.455 0.111 25 0.0182
Fract. 13 0.029 0.112 0.102 25 0.0045
Fract. 14 0.004 -0.155 NA 25 -0.00621
† Used to determine the specific activity, as shown in sample calculation for Table 2, Section C.
*SampleCalculations

3. The concentration of the undilutedextract andflow-throughwere toohightotestfor
absorbance.However,once diluted,the concentrationof the dilutedsample canbe usedtofind
the concentrationof the original sample usingthe dilutionequationarrangedassuch:
𝐶1 =
𝐶2 𝑉2
𝑉1
The concentrationof Extract iscalculatedas:
𝐶1 =
0.671
µ𝑔
µ𝑙
∗ 1000µ𝑙
5µ𝑙
𝐶1 = 134.2µ𝑔/µ𝑙
From linearregressionof the absorbance of standards(BSA),alineartrendwiththe equationy=
m (x) + b was obtained.The massof Extract/2 (x) withanabsorbance (y) of 0.332 was calculated
as such:
𝑦 = 0.0934𝑛𝑚/µ𝑔 ( 𝑥) + 0.0185
0.332𝑛𝑚 = 0.0934 ( 𝑥) + 0.0185
0.332𝑛𝑚 − 0.0185 = 0.0934 ( 𝑥)
0.313𝑛𝑚 = 0.0934𝑛𝑚/µ𝑔 ( 𝑥)
0.313𝑛𝑚 µ𝑔
0.0934𝑛𝑚
= ( 𝑥)
3.36µ𝑔 = ( 𝑥)
The Concentrationthenwascalculatedbyusingthe masscalculatedpreviously.The
concentrationof Extract/2 iscalculatedassuch:

4. 𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 =
𝑚𝑎𝑠𝑠 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒
𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 𝑎𝑑𝑑𝑒𝑑
𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 =
3.36µ𝑔
5µ𝐿
𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 = 6.71µ𝑔/µ𝐿
C. SpecificActivityof Extract and PurifiedFraction
Table 2: Determiningthe SpecificVelocity. Afterfindingthe initial velocity(O.D./min;not
shown) usingaspectrophotometer,the correctedvelocitywascalculatedbysubtractingthe
velocityof micrococcuscellsalone astheysettledtothe bottomduringthe absorbance reading.
Thiswas thenconvertedtothe velocityin (mg/mL)/min,orthe change inthe concentrationof
cellsovertime.Thiswasusedtodetermine the specificactivityof the reactionsample.Before
the specificactivitycouldbe determined,however,the total proteinconcentration(µg/mL) was
foundusingthe dilutionequationwiththe concentrationof the sample determinedinprevious
experimentswiththe BradfordAssay(Seetable 1).
Sample Velocity
(O.D./min)
Velocity
((mg/mL)/min)
Total Protein
Concentration
(µg/mL)
SpecificActivity
(mg cells/µg
lysozyme)
Extract 0.113* 0.0431** 671 *** 6.42*10-4
****
Fract. 10 0.274 0.105 0.116 0.901
Sample calculations:
*Initial velocitieswere obtainedusing a spectrophotometer,andwere correctedforthe rate of
micrococcuscellssettling.Initial Velocityof samplesandcellsusedtocorrectvelocityare not
shown,however,the correctedvelocitywascalculatedassuch:
𝐶𝑜𝑟𝑟𝑒𝑐𝑡𝑒𝑑 𝑉𝑒𝑙𝑜𝑐𝑖𝑡𝑦 𝑜𝑓 𝐸𝑥𝑡𝑟𝑎𝑐𝑡
= 𝐼𝑛𝑖𝑡𝑖𝑎𝑙 𝑉𝑒𝑙𝑜𝑐𝑖𝑡𝑦 𝑜𝑓 𝑆𝑎𝑚𝑝𝑙𝑒 − 𝑉𝑒𝑙𝑜𝑐𝑖𝑡𝑦 𝑜𝑓 𝑀𝑖𝑐𝑟𝑜𝑐𝑜𝑐𝑐𝑢𝑠 𝐶𝑒𝑙𝑙𝑠
𝐶𝑜𝑟𝑟𝑒𝑐𝑡𝑒𝑑 𝑉𝑒𝑙𝑜𝑐𝑖𝑡𝑦 𝑜𝑓 𝐸𝑥𝑡𝑟𝑎𝑐𝑡 = 0.114 − 0.001
𝐶𝑜𝑟𝑟𝑒𝑐𝑡𝑒𝑑 𝑉𝑒𝑙𝑜𝑐𝑖𝑡𝑦 𝑜𝑓 𝐸𝑥𝑡𝑟𝑎𝑐𝑡 = 0.113

5. **
Δ[cells]
min
= (
𝑉𝑒𝑙𝑜𝑐𝑖𝑡𝑦 𝑜𝑓 𝑆𝑎𝑚𝑝𝑙𝑒(𝐸𝑥𝑡𝑟𝑎𝑐𝑡)
𝑏
)
Δ[cells]
Δ𝑡
= (
0.113
𝑂. 𝐷.
𝑚𝑖𝑛
2062
𝑚𝑔
𝑚𝐿
)
Δ[cells]
Δt
= (
0.113
𝑂. 𝐷.
𝑚𝑖𝑛
2062
𝑚𝑔
𝑚𝐿
)
Δ[cells]
Δt
=
0.0431
𝑚𝑔
𝑚𝑙
𝑚𝑖𝑛
***
𝐶1 𝑉1 = 𝐶2 𝑉2
(134.2
µ𝑔
µ𝐿
) (5µ𝐿) = 𝐶2(1000µ𝐿)
(134.2
µ𝑔
µ𝐿
†
)(5µ𝐿)
1000µ𝐿
= 𝐶2
0.671
µ𝑔
µ𝐿
= 𝐶2
†ObtainedfromTable 1
To convertto µg/mL:
0.671
µ𝑔
µ𝐿
∗
1000µ𝐿
1𝑚𝐿
= 671
µ𝑔
𝑚𝐿
****
𝑆𝑝𝑒𝑐. 𝐴𝑐𝑡𝑖𝑣𝑖𝑡𝑦 =
Δ[cells]
Δ𝑡
𝑅𝑒𝑎𝑐𝑡𝑖𝑜𝑛 𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 (
µ𝑔
𝑚𝐿
)
𝑆𝑝𝑒𝑐. 𝐴𝑐𝑡𝑖𝑣𝑖𝑡𝑦 =
0.0431
𝑚𝑔
𝑚𝑙
𝑚𝑖𝑛
671
µ𝑔
𝑚𝐿
𝑆𝑝𝑒𝑐. 𝐴𝑐𝑡𝑖𝑣𝑖𝑡𝑦 = 6.42 ∗ 10−4
𝑚𝑔 (𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 𝑙𝑦𝑠𝑒𝑑)
µ𝐿 (𝑜𝑓 𝑙𝑦𝑠𝑜𝑧𝑦𝑚𝑒)
𝐃. SDS-PAGEAnalysisof Lysozyme Purification

6. Figure 2. SDS-PAGEanalysisof Lysozyme Fractions showsthe gel containingproteinstandard
(PS),eggextract(EE),flow-through(FT),andlysozymefractions10,11, 12, and13. Withinthe
lane of proteinstandard,BSA and Trios.Isomerase are markedtothe leftbya redand blue
asterisk(*) respectively. Theirlocationisknownastheyhave adoubledintensitytoserve as
reference points.Theirlocationindicatesthatlysozyme islocatedatthe pointindicatedbythe
greenasterisk.
Discussion
In the firstpart of a seriesof experiments,lysozyme was purifiedfromthe whitesof chicken
eggsusingcationexchange columnchromatography. Eggwhiteswere separatedfromyolk,and
underwentaseriesof filtrationandcentrifugationsteps.The final supernatantcontainedthe egg
extract.A 1mL sample wassetaside forcomparisonof purifiedlysozyme samples.The extractwas
pouredthroughthe columncontainingnegativelycharged,wherepositivelychargedmolecules,
includinglysozyme,became boundtothe resin.Usingalow-saltbuffer, anyunchargedornegatively
chargedmoleculeswouldrunthroughthe column.Thisrepresentedthe flow-through,andwascollected
whencolumnoutflowbegantoincrease inabsorbance.In figure 1,the flow-throughisrepresentedby
the blue diamondsandbeginsata highabsorbance reading.The volume isthe total volume of outflow
collected,startingfromthe momentanincrease inabsorbance wasmeasured.Slowly,itisshowninthe
figure thatthe absorbance drops,and thena drasticdecrease isseenafter5mL of flow-throughwas
collected.The absorbance remainedrelativelythe same overthe nextfew readings,anditwas
determinedthatall negativelychargedandunchargedmoleculeswere elutedfromthe column. This
endedthe collectionof flow-through,andthusbeganthe collectionof purifiedlysozyme fractions. At
thispoint,anypositivelychargedmoleculeswere thenelutedthroughincreasingsaltconcentrations.
Lysozyme wasexpectedtohave the greatestpositive charge andwasexpectedtoelute last.As the salt
concentrationof the bufferincreased,the outflowof the columnwascollectedin3mL fractionsand the
*
*
*

7. absorbance foreach fractionwastested.Everyotherfractioncollectedwastestedforlysozymeactivity
by an assay.A cloudysuspensionof micrococcuscellswere mixedwithasample of the fractions
collected(everyotherfractionwastested,ex.Fractions1,3, 5, etc.) to determine if the fraction
containedlysozyme.The assaywaspositive forlysozyme withinaspecificfractionif the cloudy
suspensioncleared.Those thatproducedapositive resultare showninthe redbox inFigure 1. The
lysozyme activitywasobservedinfractionsof saltconcentrationscorrespondingtoaconcentrationof
0.5M-0.7M on the graph. Atthispoint,lysozyme presence wasknown,howeveritspuritywasstill tobe
determined.
UsingSDS, we were able todetermine the purityof the eggextract,flow-through,andvarious
fractionsbelievedtocontainlysozyme.In figure 2,the proteinmarkerinthe firstlane at the leftendof
the gel was usedtodetermine whichsamplescontainedlysozyme.Accordingthe proteinstandard,
lysozyme shouldbe the secondband below Triosephosphate isomerase,indicatedbythe lowerof the
twohighintensitybands.In figure 2, the locationof Trios.Isomerase isindicatedbyablue asterisk,
while the bandwhichcorrespondstolysozymeisindicatedbya greenasterisk. Whencomparedtothe
otherlanes, itisshownthat eachfractionhad a highconcentrationof the enzyme. Thatthislane
representslysozyme isalsosupportedby figure 1,whichshowsthat fraction10 had the highest
absorbance readingandthusthe greatestconcentrationof protein. Itislogical toconclude thatwe
mightsee greaterintensityof lysozymeandotherimpurities,whichisindeedthe case.
Interestingly,the eggextractshouldhave emittedabandfor lysozyme whereasthe flow
throughshouldnot.A bandcorrespondingtolysozyme appears,thoughlight,inboth.Withinthe egg
extract,the concentrationof lysozyme maybe toolow due tothe presence of otherproteins,causing
lysozyme totake upa faintband. The flow-throughappearstohave similarbandswhencomparedto
the egg extract,butwithgreaterintensity.Thisismostlikelybecause the onlydifferencebetweenthe
twosamplesisthe presence of lysozyme ineggextract,whereasit shouldbe absentinthe flow-through.
Althoughthere issome lysozyme presentwithinthe flow-through,itmusthave alesserconcentration
comparedto the extract.Thiswouldmean otherproteinshadmade upa greaterconcentrationof the
solutionandthustookup a darkerstain whencomparedtothe extract.
As faras purityof the fractions,we can see from figure 2 thatthe fractionsare impure,
containingatleastan additional protein.Eachfractioncontainedabandwhichappearsto correspondto
aprotinin.These bandsappearabsentinthe eggextractandflow-through,suggestingthatthe column
was noteffective inseparatingall non-lysozyme proteins.Aprotininismostlikelysimilarincharge to
lysozyme,orperhapsevenabitmore charged,causingitto be elutedatthe same time as lysozyme and
thuswas presentwithlysozymeineachfraction.Fraction10 alsohad a band correspondingtoTrypsin
inhibitor.Thiswaslikelyalso positivelycharged,andbecame stucktothe column.It wasthenelutedas
the concentrationof saltincreasedslightly.Thischarge islikelynot asstrong as lysozyme andaprotinin,
as it didnotrequire similarsaltconcentrationsolutionstoelute.
Whenlookingsolelyatthe measurementsof specificactivity,the purificationappearedtobe a
success.Thisisknownby comparingthe specificactivityof the eggextractandfraction.A solution
containingahigherconcentrationof lysozyme wouldbe expectedtohave a higherspecificactivityfor
lysozyme.The eggextractisveryconcentratedwithmultipleproteins,however,unconcentratedin
lysozyme specificallyaslysozyme isonlyafractionof the proteinsfoundwithinthe extract. Before
purification,the specificactivityof eggextractwasmeasuredtobe 6.42*10-4
mg of cellslysedperµgof
lysozyme.Afterpurification,thisnumbergreatlyincreasedto0.901 mg of cellslysedperµgof lysozyme.
Thiscorrespondstoan almost1400x increase.This increasedconcentrationisalsoobservedinfigure2

8. as discussedpreviously.Whenthe bandof lysozyme infraction10iscomparedto the bandof lysozyme
inthe eggextract,the concentrationisgreatlyincreasedinfraction10.It isthe largestbandon the
plate.The eggextracthas such a small concentrationof lysozyme onthe otherhanditisbarelyvisible
and isalmostmistakenfora mere shadow.