fdsc 4763 ch 9 - protein analysis
TRANSCRIPT
-
8/12/2019 FDSC 4763 Ch 9 - Protein Analysis
1/45
FDSC4763AnalysisofFoodProducts
-
8/12/2019 FDSC 4763 Ch 9 - Protein Analysis
2/45
-
8/12/2019 FDSC 4763 Ch 9 - Protein Analysis
3/45
Thenature
of
proteins
Proteinsserveanumberoffunctionsinplantsandanimals:
Many
types
of
structural
proteins,
e.g.
collagens,
etc. Manybiochemicallyactiveproteins,e.g.enzymes,cellwall
proteins,scaffoldingproteins,etc.
Proteinsarediverseintheirchemicalproperties.
Proteinsvary
widely
in
their
sizes
and
physical
structures.
Proteinpropertiesaredeterminedbytheiraminoacidsequence(primarystructure),theinteractionamong
aminoacids
in
local
regions
of
apeptide
chain
(secondary
structure),theoverallconformationofapeptidechain(tertiarystructure),andinsomecasestheinteractionsamongpeptidechainsinaproteinmacromolecule
(quaternarystructure).
-
8/12/2019 FDSC 4763 Ch 9 - Protein Analysis
4/45
Challengesin
analyzing
protein
contentinfoods
Thediversenatureofproteinsinfoodsmakesanalysisdifficult.
Molecular
size
varies,
measured
in
Daltons,
proteins
rangefrom~5000tomorethanamillionDaltons.
Solubilityvaries,someproteinsarereadilysolubleinsaltsolutions,someinacidsolutions,someinalcoholsolutions,andsomearehighlyinsoluble.
Presenceofnonproteinconjugatesvariesconsiderablydependingonproteinfunctionality.
-
8/12/2019 FDSC 4763 Ch 9 - Protein Analysis
5/45
Waysof
measuring
protein
content
infoods
Determinationof
total
nitrogen.
Proteinsarecomposedofaminoacidsandallaminoacidscontainnitrogen,but
Actualproteincontentvariesbasedonaminoacidprofile.
Othersourcesofnitrogenoccurinmanyfoods.
Detectionandquantificationofpeptidebonds.
Complicatedbythefactthatmolecularsizeofproteinsvaries.
Detection
and
quantification
of
aromatic
amino
acids. Complicatedbythefactthataminoacidprofilesofproteinsvary.
-
8/12/2019 FDSC 4763 Ch 9 - Protein Analysis
6/45
Waysof
measuring
protein
content
infoods(continued)
Dyebindingcapacity,ultravioletabsorptivity,and/orlightscatteringpropertiesofproteins.
Complicatedbythefactthatallofthesefactorsareinfluencedbyproteinstructure,possiblyincludingprimary,secondary,tertiary,andquaternaryproteinstructures.
-
8/12/2019 FDSC 4763 Ch 9 - Protein Analysis
7/45
Considerationsin
choosing
aproteinanalysismethod
Whatdegree
of
accuracy
is
required?
Whatdegreeofprecisionisrequired?
What
degree
of
accuracy
and/or
precision
can
we
sacrificeforspeed?
Whatdegreeofaccuracyand/orprecisioncanwe
sacrificefor
cost?
Whatdegreeofaccuracyand/orprecisioncanwesacrificeforsimplicityandeaseofanalysis?
-
8/12/2019 FDSC 4763 Ch 9 - Protein Analysis
8/45
Usesof
protein
analysis
Nutritionlabeling.
Pricing.
Thevalueofmanycommodities e.g.milk,wheat,etc.
maybe
heavily
influenced
by
protein
content.
Quantificationoffunctionalproperties.
Glutenfunctionalityinwheatflour.
Caseinfunctionalityincheesemaking.
Quantificationofbiologicalactivity.
Enzyme
activity,
e.g.
pectinases,
proteases,
trypsin inhibitors,
etc.
-
8/12/2019 FDSC 4763 Ch 9 - Protein Analysis
9/45
-
8/12/2019 FDSC 4763 Ch 9 - Protein Analysis
10/45
Kjeldahl method
Johan Kjeldahl, 1849 - 1900
http://upload.wikimedia.org/wikipedia/commons/9/95/Kjeldahl3.JPG -
8/12/2019 FDSC 4763 Ch 9 - Protein Analysis
11/45
Kjeldahl workedfor
Carlsberg
Kjeldahl first developed
his method in 1883.
As of 2009, Carlsberg
was the 4th largest
brewery group in the
world
http://upload.wikimedia.org/wikipedia/commons/4/47/Carlsberg_beer.jpghttp://en.wikipedia.org/wiki/File:Tuborg_Beer_logo.svghttp://en.wikipedia.org/wiki/File:Carlsberg_logo.svg -
8/12/2019 FDSC 4763 Ch 9 - Protein Analysis
12/45
Componentsof
the
Kjeldahl
method
TheKjeldahl method
may
be
broken
down
into
three
parts:
Digestion Organicnitrogencompoundsarereducedtoammonium.
Distillation ammoniumisconvertedtoammoniagasanddistilledoff.
Titration ammonia
gas
is
trapped
by
an
acid
solution
andthesolutionistitratedtoquantifytheamountofnitrogenpresentintheoriginalsample.
-
8/12/2019 FDSC 4763 Ch 9 - Protein Analysis
13/45
Kjeldahl digestion Digestionisthemosttimeconsumingstepintheanalysis.
Thedigestion
reaction
is
made
considerably
faster
by
use
of
catalystandaneutralsalt. Manydifferentcatalystshavebeenusedovertheyears,
includingselenium,
mercury,
and
copper.
Copper
is
usually
usednowbecauseitisrelativelynontoxic.
Inorganicsalts,suchaspotassiumsulfate(K2SO4),raisetheboilingpointofthedigestingacid.andthusthetemperature
ofthe
reaction.
The
boiling
point
of
aconcentrated
sulfuric
acidsolutionisabout330 C. TheadditionofK2SO4elevatestheboilingpointtoaround400 C.Thissignificantlydecreasesthelengthoftimerequiredfordigestion.
-
8/12/2019 FDSC 4763 Ch 9 - Protein Analysis
14/45
Kjeldahl digestion the
chemistry
The
basic
chemical
reaction
is
as
follows: OrganicN+H2SO4 (NH4)2SO4+H2O+CO2+
othersamplematrixbyproducts.
-
8/12/2019 FDSC 4763 Ch 9 - Protein Analysis
15/45
Kjeldahl distillation Thepurposeof
distillation,is
to
separatetheammoniafromthedigestionmixture.
Thisis
accomplished
by
raisingthepHofthemixtureusingsodiumhydroxide(45%NaOH
solution).This
converts
theammonium(NH4+)
ionsinthedigestionmixturetogaseous
ammonia(NH3).
-
8/12/2019 FDSC 4763 Ch 9 - Protein Analysis
16/45
Kjeldahl distillation the
chemistry
ofcapturingtheammonia
Thebasicchemicalreactionisasfollows:
NH
3+H
3B
O
3
NH
4+ +
H
2B
O
3
Notethattheabovereactionusesboricacidasthereceivingsolution. Otheracidshavebeenusedaswell.
-
8/12/2019 FDSC 4763 Ch 9 - Protein Analysis
17/45
Kjeldahl titration
Acidisaddedtotitratetheborate: H2BO3 +H+ H3BO3
Notethatthenumberofhydrogenionsconsumedisequaltothe
numberof
ammonia
gas
moleculescaptured.
-
8/12/2019 FDSC 4763 Ch 9 - Protein Analysis
18/45
Kjeldahl titrationDifferentmethodsofdetectingtheendpointareused:
Colorimetric differentindicatorsareused,includingiodine,methylred/methylblue,andindophenol.
pH pHismeasuredafterdistillationintoaknownvolumeofboricacid(musthaveboricacidinexcess).
Directmeasurementofammoniausingionchromatography.
-
8/12/2019 FDSC 4763 Ch 9 - Protein Analysis
19/45
Kjeldahl calculations
Formulafor
calculating
%
nitrogen
in
asample:
Supposeweuse0.163mlof0.01NHCl (correctedvolume)totitrateoursample,whichoriginallyweighed
2.0368
g.
What
is
our
%
N
(wet
basis)?
Ifourdrymatterinthesampleis15.39%,whatisourdry
basis
%
N?
1.12%
% N = N HCl xcorrected acid volume (ml)
sample weight (g)x 100x
14 g N
mol
7.28%
-
8/12/2019 FDSC 4763 Ch 9 - Protein Analysis
20/45
Kjeldahl calculations(cont)
NotethatKjeldahl measurestotalnitrogen.
Mustcovertnitrogentoprotein.
StandardConversionfactoris6.25
Based
on
meat,
in
which
protein
is
fairly
uniformly
16%
N
byweight.
Notethatotherconversionfactorsmaybeappropriateforotherfoods(seeTable92intext).
Usingthestandardconversionfactor,whatisour%protein(wetbasis)fromourpreviousexample?
7%
-
8/12/2019 FDSC 4763 Ch 9 - Protein Analysis
21/45
Moresample
calculations
Ifourdrybasis%proteinis27.5%andourdrybasis%
moistureis
566%,
what
is
our
wet
basis
%
nitrogen
(assumestandardconversionfactor)?
First, calculate the % dry matter in the original sample.
Recall that % moisture (dry basis) = (weight of water / weightof dry matter) x 100. Therefore, if the dry basis % moisture is
566%, then:
x / (100-x) = 5.66, where x = the portion of water in 100grams of the original sample.
Solving for x, we get x = 0.85 = 85% moisture (wet basis).
Thus, dry matter is 15% (100% - 85%) by weight in theoriginal wet sample.
-
8/12/2019 FDSC 4763 Ch 9 - Protein Analysis
22/45
Moresample
calculations
(continued)Second, convert dry basis % protein to wet basis % protein:
Because the % dry matter in the original sample was
15%, our conversion factor is (1 / 0.15) = 6.67. Toconvert, we divide the dry basis % protein by our
conversion factor.
27.5% / 6.67 = 4.1% protein (wet basis).
Third, convert wet basis % protein to wet basis % nitrogen
using the standard conversion factor:
4.1% / 6.25 = 0.7% nitrogen (wet basis).
-
8/12/2019 FDSC 4763 Ch 9 - Protein Analysis
23/45
Advantagesand
disadvantages
of
Kjeldahl method
Advantages: Verylittlesamplepreparationisneeded. Sizereductionis
typicallyallthatmaybeneeded.
Workswell
for
almost
all
food
types.
Mayberelativelyinexpensive.
Canbeveryaccurate(isofficialmethodforcrudeprotein).
Disadvantages: Measurestotalnitrogen,includingnonproteinsources.
Relativelytimeconsuming.
Reagentscan
be
corrosive
and/or
toxic.
Waste
disposal
can
be
aproblem.
-
8/12/2019 FDSC 4763 Ch 9 - Protein Analysis
24/45
-
8/12/2019 FDSC 4763 Ch 9 - Protein Analysis
25/45
Dumas(Nitrogen
Combustion)
method
Principle: Samplesareburnedinpure
oxygenat700to1000C.
Nitrogencontaining
compoundsareconvertedtoN2andnitrogenoxides.
Nitrogen
oxides
are
reduced
to
N2inacoppercolumnat~600C
Nitrogengasisassayedusing
gaschromatography
(GC).
-
8/12/2019 FDSC 4763 Ch 9 - Protein Analysis
26/45
DumasMethod
Equipment
-
8/12/2019 FDSC 4763 Ch 9 - Protein Analysis
27/45
-
8/12/2019 FDSC 4763 Ch 9 - Protein Analysis
28/45
Infraredspectroscopy
Principle:
Usesnearinfrared(NIR)ormidinfrared(MIR)radiationtodetectandquantifypeptidebondsinasample.
-
8/12/2019 FDSC 4763 Ch 9 - Protein Analysis
29/45
Advantagesand
disadvantages
of
Infraredspectroscopy
Advantages: Requiresnohazardouschemicals.
Veryrapid,typicallyunder2minutestoanalyzeasample.
Verylittle
training
required
to
use
equipment.
Specifictoprotein,notaffectedbynonproteinnitrogen.
Suitableforawiderangeofproducts.
Disadvantages: Equipmentisrelativelyexpensivetopurchaseandmaintain.
Equipmentmustbecalibratedtospecificproductsinorderto
giveaccurate
results.
-
8/12/2019 FDSC 4763 Ch 9 - Protein Analysis
30/45
Biuret MethodPrinciple:
Cupric
ions
(Cu++
)
will
complex
with
peptide
bonds
in
a
proteinmoleculeunderalkalineconditons.
Theproteincoppercomplexabsorbslightwithpeakabsorbanceoccursat540nm. (Thisgivesthesampleapurplishcolor.) Thedegreeofabsorbanceisproportionaltotheamountofproteininthesample.
-
8/12/2019 FDSC 4763 Ch 9 - Protein Analysis
31/45
Biuret Method:Procedure
Biuret reagent(solutionofcoppersulfate[CuSO4],NaOH,
and
potassium
sodium
tatrate [NaKC4H4O6])is
mixedatabouta5:1ratiowithaproteinsolution.
Mixtureisallowedtostandforapproximately15to30minutesatroomtemperature.
Absorbanceisreadat540nmusinga
spectrophotometer
and
a
Biuret reagent
blank.Proteinconcentrationiscalculatedusingastandard
curve,typicallyconstructedusingbovineserum
albumin(BSA).
-
8/12/2019 FDSC 4763 Ch 9 - Protein Analysis
32/45
Biuret methodapplications
The
Biuret Method
is
commonly
used
to
determine
proteinconcentrationin:
Cerealproducts.
Meats. Soybeanproducts.
Animalfeeds.
Proteinconcentrates
and
isolates.
-
8/12/2019 FDSC 4763 Ch 9 - Protein Analysis
33/45
Advantagesand
disadvantages
of
Biuret Method
Advantages: Simplemethod,requiresnospecializedequipment.
Relativelyrapid,typically~30minutestoanalyzeasample.
Specific
to
proteins,
not
affected
by
non
protein
nitrogen. Rarelyencounterinterferencefromothercomponents.
Disadvantages: Notparticularlysensitive.
Colormayshiftdependingonproteinaminoacidprofileandstructure.
Samplesmustberelativelyclearandnonturbid.
Mustbe
standardized
against
aknown
protein,
e.g.
results
reportedasBSAequivalents. Thisusuallyreducesaccuracy.
-
8/12/2019 FDSC 4763 Ch 9 - Protein Analysis
34/45
LowryMethod
Principle:
Combines
Biuret Methodwithadditionalreagentsandreactionstoboostsensitivity.
Thecomplexofcupricions(Cu++)withpeptidebonds
underalkaline
conditons reduces
cupric
ions
to
cuprous
ions(Cu+).
Alongwithtyrosineandtyptophan sidechains,cuprous
ions
react
to
reduce
Folin
Ciocalteu Reagent
(phosphomolybdic/phosphotungstic acid).
Thisproducesabluecolorthatcanbereadat750nm(highsensitivity)or500nm(lowsensitivity). The
amountof
absorbance
is
proportional
to
the
number
of
peptidebonds.
-
8/12/2019 FDSC 4763 Ch 9 - Protein Analysis
35/45
LowryMethod
applications
The
Lowry
Method
is
commonly
used
to
determine
proteinconcentrationin:
Isolatedcellfractions.
Chromatographyfractions.
Enzymepreparations.
Otherproductsofproteinbiochemistry.
Notgenerallyusedforcomplexfoodsystems;maybeusedonisolated,purifiedfoodproteins. Somewhat
supersededby
the
BCA
method.
-
8/12/2019 FDSC 4763 Ch 9 - Protein Analysis
36/45
Advantages
and
disadvantages
of
LowryMethod Advantages:
Simplemethod,
requires
no
specialized
equipment.
ProceduresaresimilartoBiuret Method,typically~11.5hourstoanalyzeasample.
Specific
to
proteins,
not
affected
by
non
protein
nitrogen. MethodismuchmoresensitivethanBiuret method. MethodislesssensitivetoturbiditythanBiuret method.
Disadvantages: Color
may
shift
depending
on
protein
amino
acid
profile
and
structure. MoreaffectedbyothersamplecomponentsthanBiuret
method,especiallyoxidizing/reducingagents.
Mustbe
standardized
against
aknown
protein,
e.g.
results
reportedasBSAequivalents. Thisusuallyreducesaccuracy.
-
8/12/2019 FDSC 4763 Ch 9 - Protein Analysis
37/45
DyeBinding
Methods
Principle: Dye
molecules
bind
to
proteins,
oftentoNterminalaminoacids.
Bounddyemoleculeseffecta
changein
the
solutions
absorbance
orfluorescence.
Degreeofchangeisproportional
to
the
amount
of
protein
present. Absorbanceorfluorescencecomparedtoastandard
curvetocalculateproteinconcentration.
Manydifferentspecifictestsexist,manyareintendedfortesting
specific
types
of
foods.
-
8/12/2019 FDSC 4763 Ch 9 - Protein Analysis
38/45
Anionicdye
binding
Applications: Milk;wheatflour;soyproducts;meats.
Advantages: Rapid(lessthan15minutes,typically). Relativelyaccurateandprecise.
Does
not
measure
non
nitrogen
proteins. Maybeusedspecificallytoquantitate changesinlysine
contentincerealproducts.
Disadvantages:
Notsensitive
to
less
than
milligram
quantities
of
protein.
Calibrationcurverequiredforeachtypeofsamplebeinganalyzed.
Willnotdetecthydrolyzedproteins. Non
protein
components
(e.g.
starch)
may
bind
dye
and
introduceinaccuracies.
-
8/12/2019 FDSC 4763 Ch 9 - Protein Analysis
39/45
Bradforddye
binding
UsesCoomassie BrilliantBluedye. Applications:
Worts (beerproducts).
Potatoes. Enzymeconcentrations.
Advantages: Veryrapid(~2minutes). Verysensitive. Lesssusceptibletointerferencethanmanyotherchemical
methods. Disadvantages:
Calibrationcurverequiredforeachtypeofsamplebeinganalyzed maybeveryspecifictoagivenproduct.
Proteindye
complex
can
be
sticky.
Must
use
glass
or
plastic
cuvettes foranalysis.
-
8/12/2019 FDSC 4763 Ch 9 - Protein Analysis
40/45
Bicinchoninic Acid(BCA)
Method
Principle:
Cupricionsarereducedtocuprousionsunderalkalineconditions(similar
to
Biuret reaction).
Cuprousionsreactwithbicinchoninic Acid
(BCA)
to
formacomplexthatabsorbsat562nm(purplishcolor).
BCA Molecule
-
8/12/2019 FDSC 4763 Ch 9 - Protein Analysis
41/45
BCAMethod
Uses
Applications: Oftenusedinproteinpurificationandisolation,similarto
Lowrymethod.
Not
used
for
measuring
protein
in
complex
foodsystems.
Advantages: AssensitiveasLowrymethod,butprocedureissimpler.
ReagentismorestablethanLowrymethod.
Relativelylesssusceptibletointerferencefromnonproteincomponents.
Disadvantages: Colorofreactionproductsisnotstablewithtime.
Reducingcompoundswillinterferewiththetest.
Colorof
reaction
products
will
vary
according
to
protein
structure,similartoLowryMethod.
-
8/12/2019 FDSC 4763 Ch 9 - Protein Analysis
42/45
-
8/12/2019 FDSC 4763 Ch 9 - Protein Analysis
43/45
UVAbsorption
applications
TheUV
Absorption
Method
is
commonly
used
to
determineproteinconcentrationin:
Milk.
Meat. Purifiedproteinpreparations.
Notoften
used
for
complex
food
systems
containing
proteinswithdifferingsolubilitycharacteristics.
-
8/12/2019 FDSC 4763 Ch 9 - Protein Analysis
44/45
UVAbsorption
advantages
and
disadvantages
Advantages: Veryrapid(onceproteinissolubilized). Nointerferencefromothercommonnitrogencontaining
components,e.g.ammoniumsulfateandotherbuffering
agents. Nondestructive preparedproteinsolutionscanbeusedforotherpurposesafterUVabsorbanceismeasured.
Disadvantages:
Nucleicacids
interfere
with
the
test
as
they
also
absorb
at
280
nm. Proportionsoftyrosineandtryptophancanvaryamong
differentfoodproteins,calibrationmayberequired.
Proteinsolutions
must
be
clear,
colorless,
and
fairly
pure
for
themethodtobeaccurate.
-
8/12/2019 FDSC 4763 Ch 9 - Protein Analysis
45/45
Proteinanalysis
final
thoughts
Manydifferentmethodsofproteinanalysisexist
mustmatch
method
to
foodstuff
and
be
aware
of
the
limitationofanygivenmethod.
Methodsthatmeasuretotalnitrogen(e.g.Kjeldahl andDumas)
will
usually
overestimate
crude
protein
content.
Methodsthatmeasureproteindirectlywillusuallygivedifferentresultswithdifferenttypesofproteins proper
calibrationis
amust.
Measurementsofcrudeproteinwillusuallynotbesufficienttodetermineactualproteinnutritivevalue,e.g.
Protein
Efficiency
Ratio
(PER).