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ANALISIS PROTEIN---------------------------------------------

Disampaikan olehSri Naruki

Fakultas Teknologi Pertanian UGM

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PROTEIN

• Protein are an abundant component inall cells (Nielsen, !!"#• Food protein $ %er& comple' M)

ranging *rom +!!! to more than amillion Daltons

• Function $ gro th maintenance o*tissue, *ormation o* essential bod&

compounds, transportation o* nutrients,etc. (Guthrie, /01 #

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• Protein consist o* pol&peptides,

e'tremel& long chains o* man& aminoacid units• 2ll protein in all species, regardless o*

their *unction or biological acti%it& ,are built *rom the same basic set o*

! standard amino acids, hich b&themsel%es 3245 N6 intrincsicbiological acti%it&

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• General structure o* amino acids $ 7663

3 N−7 −3 8

8 9 side chain or 8 group

• The simples amino acid $ gl&cine → 8 9 3• :n all amino acids, e'cept gl&cine, the

carbon atom has " di;erent substituent

groups and is thus as&metric or chiralcarbon (

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The Peptide >ond

• Peptide bond $ ho the amino acidsare linked together to make a protein 3 H

3 2 N77663 ? H 2 NCCOOH 8 R’ 3 6 H

3 2 N-7-7- N-C-COOH ? 3 2 6 8 H R’

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• Proteins are composed o* 3 , 7 , N, 6 ,and S (Nielsen, !!"#

• Composition of elements inproteins :

- 7 $ +! @ ++ A- 6 $ ! @ + A- N $ /B." @ /0./ A

- 3 $ + @ C A- S $ !." @ .+ A- P, Fe, 7u $ trace

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• C (karbon) : merupakan penyusun terbesar • Bila C digunakan sebagai dasar analisis

protein• Analisis protein berdasarkan C

mempunyai beberapa keuntungan :

a. Digesti lebih mudah bila dibandingkandengan digesti pada metoda analisisberdasarkan N

b. Kadar C protein = tinggi sehingga- mengurangi error- aktor kon!ersi relati konstan

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• "A#$ $N%A" : ada beberapa senya&alain yang 'uga mengandung C →

karbohidrat dan lipida• leh karena itu C nonprotein tersebut

harus dihilangkan terlebih dahulu

• #emisahan C nonprotein tersebut → sulit

leh karena itu yang la*im dilakukanadalah : penentuan kadar proteinberda-sarkan kadar nitrogen

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• Nitrogen is the most distinguishing

element present in proteins• N content in %arious *ood protein ranges

*rom /B."A to /0./A → an a%erage 9/=.!A

• There*ore, protein content (A#9 {/!! /= }' A N9 6.2 ' A N

con%ersion *actor C! (*aktorkon%ersi !" #

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• N content in %arious *ood protein ranges

*rom /B."A to /0./A → con%ersion*actors *or %arious *ood are %aried $- 5gg or meat → C! 9 =. +- Dair& products =.B1- )heat +.C!- 6ther cereal grains oilseeds

=. +- 2lmonds +./1- Peanuts >raEil nuts +."=- 6ther tree nuts coconut +.B!

Con#ersion !$%tors

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• 2nal&sis o* protein is %er& complicatedsince some *ood components possessimilar ph&sicochemical properties

• Nonprotein nitrogen could come *rom $- Free amino acids - Some %itamins- Small peptide - 2lkaloids

- Nucleic acids - Uric acid- Porph&rin - Urea- 2mmonium ions - 5tc

ANAL&SIS O! PROTEIN

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2spartame is a sm$ll pepti'e composed o*2sp, Phe, and methanol

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Nucleic acids are polymer formed from linking ofvarious nucleotides

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Struktur vitamin B 1 (tiamin) → juga mengandung N

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7a;eine $l($loi'

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7hemical structure o* )ri% $%i'

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7hemical structure o* )re$

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• The total organic nitrogen in *oods ouldrepresent nitrogen $

- Primaril& *rom protein- 2 lesser e'tent *rom all organic nitrogen-containing nonprotein substances

• There*ore, A Protein 9 7F ' A N is called $ crude protein ( protein kasar #• 6ther ma or *ood components, including

carboh&drate and lipid, ma& inter*ere

ph&sicall& ith anal&sis o* *ood proteins

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/. Nutrition labeling. Functional propert& in%estigation

Proteins o* *ood ha%e uni ue *unctionalproperties. 5'ample $ gliadin gluteninin heat Hour *or bread making

B. >iological acti%it& determination, including enE&mes enE&me inhibitor. 5nE&me

acti%it& is e'pressed in terms o* speciIcacti%it& $ unit o* enE&me mg o* protein

Import$n%e of An$l*sis

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• Protein anal&sis is re uired i* &ou antto kno $

/.Total protein content.7ontent o* particular protein in mi'ture

B.Protein content during isolation and

puriIcation o* protein". Nonprotein nitrogen (e'. TM2 content#+.2mino acid composition ( pro+l asam

amino #=.Nutriti%e %alue o* protein (e'. P58, >4#C.2cti%it& (o* enE&me#

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Protein Content of Sele%te' !oo's !oo' item + Protein

, et eig t

/$sis075852

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• Determination o* nitrogen, peptidebonds, aromatic amino acids

• >ased on the d&e-binding capacit& o*

protein• >ased on the U4 absorpti%it& o*

protein

• >ased on the light scatteringproperties o* protein

T e

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The >asic Principles o* SomeMethods to Measure Protein

7ontentI. =etermin$tion of nitrogen1pepti'e /on's1 $rom$ti% $mino

$%i's

2. K eldahl method D. Titrasi *ormol>.>iuret method 5. Dumas

method7. 72 method

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A. ">EL=AHL

ETHO=

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• 6n March C, /11B, Lohann K eldahlpresented his method o* N anal&sis

• Toda&, the K eldahl method *or thedetermination o* organic nitrogen is the

orld ide standard *or the purpose o*

calculating the protein content in bothhuman *ood and animal *eed• The K eldahl method has been adapted

as a standard method o* N anal&sis in*ertiliEer, *ossil *uel, aste ater, etc.

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• Proteins other organic *ood

components in a sample are digested ith sul*uric acid in the presence o*

catal&sts• The total organic nitrogen is con%erted to

ammonium sul*ate• The digest is neutraliEed ith alkali and

distilled into a boric acid solution• The borate anions *ormed are titrated

ith standardiEed acid, hich iscon%erted to N in the sample

PRINCIPLE

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• The result o* anal&sis represents the

crude protein content o* the *ood since Nalso comes *rom nonprotein components• The K eldahl method *or nitrogen

anal&sis is composed o* three distinctsteps $

/.Digestion ( destruksi #.NeutraliEation and Distillation

B.Titration

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/. Katalis logam seperti 3g, 7u, dan Se ditam-

bahkan ke asam sul*at untuk men&empurna-kan oksidasi.- Jang terbaik $ 3g

- Selenium dioksida dan 7u-sul*at (B$/# ugae*ekti* untuk digesti

- Copper and titanium dio,ide also ha!ebeen used as a mi,ed atalyst or digestion→ the use o them poses less sa ety on ern than g in the post-analysis disposal o the&aste

I PRO?E ENTS

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• Bila digunakan Hg selama digestiakan terbentuk senya&a kompleks g-amonia

• g harus diendapkan (sebagai g/)dengan penambahan Na-tiosul at

(Na 0 / 0 1 ) → presipitan Na 0 / 0 1di ampur dengan Na yangdigunakan untuk membebas-kan N 1(saat tahap netralisasi sebelum distilasi)

• Na-tiosul at 'uga ditambahkan untukmembantu membebaskan N dariikatan-nya dengan g (lihat butir 1)

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• Di lapangan ampuran (Na 2Na 0 / 0 1 ) dikenal dengan istilah Na -

thio

• Katalis Se (selenium) :

- e ek lebih hebat daripada g- "idak perlu step tambahan (yaitu

penam-bahan Na-tiosul at 3Na 0 / 0 1 )

- "A#$ : bila 'umlah /e berlebihan makasuhu destruksi3digesti tidak terkontrolsehingga ada kemungkinan kehilanganN (karena menguap)

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. #enambahan potasium sul at dapatdila-kukan untuk menaikkan titik didih

asam sul at sehingga meningkatkandigesti/uhu digesti yang ideal : 145 6 785 oC.

"A#$ bila K-sul at berlebihan maka akanter'adi kehilangan NB. SulIde or sodium thiosul*ate are added

to the diluted digest to help releasenitrogen *rom mercur&, hich tends tobind to ammonium

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". The ammonia is distilled directl& into

a boric acid solution, *ollo ed b&titration ith standard acid+. 7olorimetr& NessleriEation, or ion

chroma- tograph& to measureammonia, is used to determinenitrogen content a*ter digestion

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Beberapa Modifkasi

1. Cara makro Kjeldahl- Berat sampel : 8 g- Katalis : K 0 / 7- g

- Distilasi : memapak lempeng 9ndengan tu'uan agar supaya

tidak ter'adi superheating

tidak ter'adi per ikan airan tidak terbentuk gelembung gas

yang besar

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@ENERAL PROCE= RE B

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@ENERAL PROCE= RE BREACTIONS

• 2s stated be*ore, the K eldahl method *or

nitrogen anal&sis is composed o* threedistinct steps. Sample preparation shouldbe done prior to these three steps.

/. Sample preparation. Digestion ( destruksi #

B. NeutraliEation and Distillation". Titration

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• Solid *ood are ground to pass a !mesh screen

• Samples *or anal&sis should be

homogeneous• No other special preparation are

re uired

. S$mple Prep$r$tion

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2. =igestion Step

• The purpose o* digestion $ to breakthe intricate structure and chemicalbonds that hold a chemicalsubstances (piece o* meat, cup o*Hour, or uart o* oil# do n into simplechemicals and ionic structures

• SpeciIcall&, proteins and other *ormso* nitrogen are broken do n andcon%erted to ammonia

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• To digest the sample, / @ g o* thesample are placed on a digestion tube

ith / @ /+ m< o* concentrated 3 S6 " .Se%en grams o* K S6 " and a metalliccatal&st, usuall& 7u, are the added.

• The digestion tube is heated to theboiling temperature o* the mi'ture

• Digestion as done to completeo'idation and con%ersion o* N toammonium sul*ate

• The digestion is usuall& completed a*terone hour at BC! @ "!! o7

$ ) f

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$. Pro%e')re of=igestion• Place sample (accuratel& eighed# in a

K eldahl Hask• 2dd sul*uric acid and catal&st

• Digest until 7

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• Non%olatile ammonium sul*ate is*ormed *rom nitrogen and sul*uricacid $

Sul*uric acid Protein (N3 " # S6 " 3eat, catal&st

• During digestion $- protein nitrogen is liberated to *orm

ammonium ions

/. Re$%tions =)ring=igestion

S l* i id 'idiE d i

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- Sul*uric acid o'idiEed organic matter combines ith ammonium *ormed

- 7 is con%erted to 76 (%olatile menguap # - 3 is con%erted to 3 6 (%olatile menguap #•

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3g6 ? 3 S6 " → 3gS6 " ? 3 6 3gS6 " → 3g S6 " ? S6 ? 6 n

(736N# ? 6 n ? 3 S6 " → 76 ? 3 6 ?(N3 " # S6 "

protein

%. eaksi lengkap

7 N )t $li $ti B

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• Pada tahap ini (N3 " # S6 " dipecah men adiN3 B dengan penambahan Na63 (sampaidica-pai kondisi alkalis# dan pemanasan(distilasi#

• Selama distilasi dapat ditambahkan lempengn• N3 B &ang dibebaskan ditangkap oleh larutan

asam standar berlebihan. 2sam dapat

berupa- 2sam borat "A- 37l !,/N

7. Ne)tr$li $tion B=istill$tion Step

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• U ung alat distilasi harus tercelup larutanasam

• Distilasi diakhiri bila destilat &ang dihasil-kan tidak lagi bereaksi basa. 7aran&a

• 8eaksi &ang ter adi saat netralisasi dandistilasi $

(N3 " # S6 " ? Na63 → N3 B ? Na S6 " ?3 6

• Selan utn&a N3 B &ang dibebaskan akan

bereaksi dengan larutan penampungsebagai berikut $

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• >ila penampungn&a asam borat $

N3 B ? 3 B>6 B ( boric acid # → N3 " ?3 >6 B@(borate ion #

• >ila penampungn&a 37l $ N3 B ? 37l ( berlebihan # → N3 " 7l ?

37l ( sisa #

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• The digest is diluted ith ater• 2lkali-containing sodium thiosul*ate

is added to neutraliEe the sul*uricacid

• The ammonia *ormed is distilled intoa boric acid solution containing theindicator meth&lene blue and meth&lred

Pro%e')re of Netr$li $tion B=istill$tion

$

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$.

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• 2 reagent blank should be run to subtractreagent nitrogen *rom the sample nitrogen

• A N 9N 37l ' (corrected acid %olume g o* sample#

' (/"g N mol# ' /!!

)here $- N 37l 9 normalit& o* 37l, in moles /!!!ml- 7orrected acid %olume 9

(ml std acid *or sample# @ (ml std acid*or blank#

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• 2tau $

A N 9 {(ml 37l sampel @ ml 37l blanko # $ beratsampel (g# ' /!!! } ' N 37l ' /",!!1 ' /!!A

• Untuk menghitung kadar protein, maka A N

dikalikan *aktor kon%ersi (7F atau FK# ↓7F beberapa enis bahan makanan $ lihat slide

sebelumn&a

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• >ila digunakan penampung 37l, maka 37lsisa dititrasi dengan Na63 standar (!,/N#

• 8eaksi- Saat distilasi $ N3 B ? 37l ( berlebihan # → N3 " 7l ?

37l ( sisa #- Saat titrasi $37l ( sisa # ? Na63 → Na7l ? 3 6

/.

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• 2tau $

AN 9 {(ml Na63 blanko @ ml Na63sampel # $ berat sampel (g# '/!!! } ' N Na63 ' /",!!1 ' /!!A

• 2 *actor is used to con%ert A N to Acrude protein

• Most proteins contain /=A N, so thecon%ersion *actor (7F# is =. +A Protein 9 A N ' =. +

PROSE= R LEN@"AP

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PROSE= R LEN@ APETO=A ">EL=AHL

!,C @ . g sampel (dalam labu K eldahl# !,Cg 3g6 (atau !,=+g logam

3g#

/+g K S6 " (atau Na S6 " anhidrat#+ml 3 S6 " pekat

=ESTR "SID G'S"# (didihkan# pelan-pelan, tambahkan sedikitparaIn

Setelah ernih, didihkan lagi ? B! menitPEN=IN@INAN (ne't slide#

P5ND:NG:N2N

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P5ND:NG:N2N ? !! ml a uades dingin

>utiran n

+ml larutan Na S 6 B 1A ? /+g Na63PE ASAN@AN RAN@"AIAN =ISTILASIPE ASAN@AN ERLEN E&ER PENA P N@

(/+ml 37l !,! N ? = tetes M8->7G#=ISTILASI (Diakhiri bila distilat mencapai O

/+!ml # (7uci u ung kondenser#TITRASI 37l sisa dalam 5rlenme&er dengan

Na63

!K K!% *'%'%" !% B !%K$ ++

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P5ND:NG:N2N1 @ /!ml Na63-Na S 6 B

=ISTILASI - Tampung distilat dalam 5rlenme&er berisi $

+ml 3 B>6 B "A ? " tetes indikator (!, A M8 ?!, A >7G#

- Distilasi diakhiri saat distilat sudah tidaklagi alkalis

TITRASI - Distilat dititrasi dengan 37l !,! N

!K K!% *'%'%" !% B !%K$ ++

ALTERNATE

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• :n place o* distillation titration ithacid, ammonia or nitrogen can be

uantitated b& $/.N5SS

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• N3 3g :6 (red-orange # can be determinedspectrophotometricall& at ""!nm

• This method is rapid sensiti%e, but theammonium dimercuric iodide is colloidal andcolor is not stable

63 @. N3 B ? phenol ? h&pochlorite

indophenol (blue, =B!nm#B. p3 measurement a*ter distillation into

kno n %olume o* boric acid

". Direct measurement o* ammonia, using ionchromatographic method

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/. 2pplicable to all t&pes o* *oods. :ne'pensi%e(i* not usingan

automated s&stem#

B. 2ccurate an o cial method *orcrude protein content#". 3as been modiIed (micro K eldahl

method# to measure microgramuantities o* protein#

2D42NT2G5S

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/. Measures total organic nitrogen, not ust protein nitrogen

. Time consuming (at least hr tocomplete#

B. Poorer precision than the >iuretmethod

". 7orrosi%e reagent

D:S2D42NT2G5S

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76NT63 S62<

• Pada analisa kadar protein denganmetoda K eldahl, diperoleh hasil kadarprotein susu bubuk skim adalah "!A.Penampung distilat &ang digunakanadalah 37l berlebihan. >ila berat sampel9 !, +g dan titrasi blanko membutuhkanNa63 !,/N seban&ak "+ml, hitunglah

%olume Na63 untuk titrasi sampel. Faktorkon%ersi N ke protein skim 9 =,B1.

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>. >:U85T M5T36D

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• 2 %iolet-purple color is produced hencupric ions are comple'es ith O pep-tide bonds, under alkaline

conditions#• The absorbance o* the color produced

is read at +"! nm

• The color intensit& (absorbance# isproportional to the protein content o*the sample

/. P8:N7:P

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3 6 3366@7@N@7 @7 @N3 ? 7uS6 " ? Na63 → 8 3 8 8 3 3 8

3N@7@7@N@7@7663 3 6 3 7u ? Na S6 " ? 3 6 3 6

366@7@N@7@7@N3 8 3 3 8 (sen&a a kompleks ungu#

. 8527T:6NS

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" 2D42NT2G5S

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•

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/. Kurang sensiti* bila dibandingkandengan metoda

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". )arna &ang dihasilkan ber%ariasidengan perbedaan enis protein. Misal $

gelatin menghasilkan arna pinkish-purple+. Kondisi Rtak tembus caha&a dapat

ter adi pada larutan Inal apabilaterdapat lipida atau karbohidrat dalam

umlah ban&ak=. >ukan merupakan metoda &ang absolut

$ arna harus distandardisasi denganprotein &ang diketahui (misal >S2# ataudicocokkan dengan metoda K eldahl

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3ubungan antara absorbansi pada+"! nm pada >iuret method dengan

kadar protein menurut metodaK eldahl

2 a

t + " ! n m

A Protein (K eldalh#

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7.

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• The # ith thereduction o* the Folin-7iocalteauphenol reagent ( phosphomol&bdic-

phosphotungstic acid # b& t&rosin tr&ptophan residues in the protein• The bluish color de%eloped is read at

C+! nm (high sensiti%it& *or lo proteinconcentration# or +!! nm (losensiti%it& *or high proteinconcentration#

/. P8:N7:P

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Protein reaction with cupric ion under

alkaline condition

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• Terdapat macam reagen

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• 7ara $- / m< larutan protein sampel ? +m<

reagen , go og dan biarkan/! menit

- Kemudian tambahkan !,+ m< reagen

aca nilai absorbansi pada =!! nm

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/. Proteins to be anal&Eed are diluted toan appropriate range ( ! @ /!! µg#. K Na Tartrate-Na 76 B solution is

added a*ter cooling and incubated atroom temperature *or /! minB. 7uS6 " -K Na Tartrate-Na63 solution is

added a*ter cooling and incubated atroom temperature *or /! min

b. P8675DU85

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C ++ i lk li l ti t f l it ith

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• Cu ++ in alkaline solution to form complexity with protein.

• Cu ++ catalyses oxidation of phenol group of tyrosinewith phosphomolybdic-phosphotungstic acid.

2 a

t C + ! n m

g o* protein (K eldahl#µ

B. 2D42NT2G5S

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/. 4er& sensiti%e $a. +! @ /!! times more sensiti%e than biuret

methodb. /! @ ! times more sensiti%e than 1!nm

U4 absorption method

c. Similar sensiti%it& as NessleriEationho e%er, more con%enient thanNessleriEation

.

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/. 7olor %aries ith di;erent proteins to a

greater e'tent than biuret method. 7olor is not strictl& proportional to

protein concentration

B. The reaction is inter*ered ith to%ar&ing degrees b& sucrose, lipids,phosphate bu;ers, monosaccharides,and he'oamines

". 3igh concentration o* reducing sugars,ammonium sul*ate, and sul*h&dr&lcompounds inter*ere ith the reaction

. D:S2D42NT2G5S

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D. T:T82S: F68M6<

8

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•

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• :ndikator &ang dipakai adalah PP• 2khir titrasi ditandai saat ter adin&a

perubahan arna men adi merah muda&ang tidak hilang dalam B! detik

• Titrasi *ormol baik untuk digunakan

untuk e%aluasi proses ter adin&apemecahan protein (misal $ pada*ermentasi protein pada tempe, kecap,

tauco, dEsb.#• Proses hidrolisis protein ditandaidengan meningkatn&a titrasi *ormol

. 852KS: P2D2 T:T82S:

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6 3 68@73@7@63 ? Na63 → 8@7@7@6 N3 N3 BF

pada p3 netral 3 6 3 8@7@7@6 ? 73 6 → 8@7@7663 N3 BF ( *ormalin # 363 7@N@73 63 (dimetilol # 3 8@7@7663 3 6363 7@N@73 63 ? Na63 → 8@7@7@6Na

(dimetilol # 363 7@N@73 63

F68M6<

B. P86S5DU8

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• /! ml larutan protein sampel ? ! ml a u-ades ? !," ml larutan K-oksalat enuh (K-oksalat $ air 9 / $ B# dan / ml indikator PP/A. Diamkan selama menit

• Titrasi larutan tersebut dengan !,/N

Na63 sampai terbentuk arna pink atauarna standar (/! ml susu ? /! ml

a uades ? !," ml K-oksalat enuh ? /tetes indikator rosanilin-khlorida !,!/A#

• Setelah arna tercapai tambahkan ml

8/18/2019 PT. 7. Protein

92/125

Setelah arna tercapai, tambahkan ml*ormaldehid "!A dan titrasilah kembalidengan larutan Na63 sampai arnaseperti arna standar tercapai lagi.7atatlah nilai titrasi kedua ini.

• Dibuat titrasi blanko &ang terdiri dari $! ml a uades ? !," ml larutan K-

oksalat enuh ? / ml indikator PP ? ml*ormal-dehid, dan titrasilah dengan

larutan Na63• Titrasi *ormol 9 titrasi terkoreksi 9titrasi kedua @ titrasi blanko

• >ila nilai titrasi *ormol akan digunakan

8/18/2019 PT. 7. Protein

93/125

guntuk menentukan kadar protein, makaharus dibuat percobaan serupa denganmenggunakan larutan &ang telahdiketahui kadar proteinn&a (misaln&adengan metoda K eldahl#

• Selan utn&a ditentukan hubunganantara titrasi *ormol dengan A protein.

• Misal $

- A protein susu 9 /,1B ' ml titrasi *ormol- A kasein 9 /,=B ' ml titrasi *ormol

8/18/2019 PT. 7. Protein

94/125

5. DUM2S(N:T86G5N

76M>UST:6N#M5T36D

/ P8 N7 P

8/18/2019 PT. 7. Protein

95/125

• Sample are combusted at high tempera-ture ( !! " #$!!! o %)

• &he nitrogen released is 'uantitated bygas chromatography using thermalconductivity detector (&% )

• &he nitrogen determined is converted toprotein content in the sample

/. P8:N7:P

8/18/2019 PT. 7. Protein

96/125

B. 2D42NT2G5S D S2D42NT2G5S

8/18/2019 PT. 7. Protein

97/125

. 2.N&.3+S

• e'uires no ha4ardous chemicals• %an be accomplished in 5 minutes• ecent automated instrument can analy4e

up to # ! samples without attention0S. 2.N&.3+S

• +6pensive e'uipment is re'uired• 7easures total organic nitrogen8 not just

protein nitrogen

D:S2D42NT2G5S

8/18/2019 PT. 7. Protein

98/125

F. >:7:N736N:N:7

27:D (>72# M5T36D

/ P8 NS P

8/18/2019 PT. 7. Protein

99/125

• Protein mampu mereduksi ion kupri (7u ? #men adi ion kupro dalam suasana alkalis

• Dengan reagen >72 (ber arna apple-

greenish #, ion kupro tersebut membentukkompleks ber arna purplish , &angintensitasn&a dapat ditera pada += nm

• :ntensitas arna purplish tersebut propor-sional dengan kadar protein

/. P8:NS:P

8/18/2019 PT. 7. Protein

100/125

8/18/2019 PT. 7. Protein

101/125

P86S5DU8

8/18/2019 PT. 7. Protein

102/125

• Mi' (one step# the protein solution ith

the >72 reagent, hich contain >72sodium salt, Na-carbonate, Na63, and7u-sul*ate p3 //. +

• :ncubate at room temperature *or hr,or =! o7 *or B! min. 2 highertemperature gi%es a greater colorrespon

• 8ead the solution at += nm against areagent blank

• 7onstruct a standard cur%e using >S2

. P86S5DU8

B 2PP

8/18/2019 PT. 7. Protein

103/125

• >72 method had been used inprotein isolation and puriIcation

• The suitabilit& o* >72 method *ormeasuring protein in comple' *oodhas not been reported

B. 2PP

8/18/2019 PT. 7. Protein

104/125

/. Sensiti%it& o* the micro >72 method(!.+ @ /! µg# is better than

8/18/2019 PT. 7. Protein

105/125

/. 7olor is not stable ith time. Theanal&st needs to care*ull& control thetime *or reading absorbance

. 2n& compound capable o* reducing7u ? to 7u ? ill lead to color *ormation

B. 8educing sugar inter*ere to a greatere'tent than in

8/18/2019 PT. 7. Protein

106/125

II.

8/18/2019 PT. 7. Protein

107/125

/ P8:N7:P

8/18/2019 PT. 7. Protein

108/125

• Protein sho strong absorption at 1!nm, primaril& due to t&rosine (T&r#and tr&ptophan (Trp# residues in the

protein• >ecause the content o* T&r Trp in

protein *rom each *ood source is *airl&

constant, the 2 1! could be used toestimate the concentration o* protein

/. P8:N7:P

8/18/2019 PT. 7. Protein

109/125

• Protein are solubiliEed in bu;er or alkali

• 2bsorbance o* protein solution is readat 1! nm against a reagent blank

• Protein concentration is calculated

according >eer s la $ A = ab• 2 9 absorbance, a 9 konstanta (molar

absorp- ti%it& *or indi%idual protein#, b 9

cu%ette path length, c 9 concentration• >isa uga memakai standar >S2

. 86 5DU85

B 2PP

8/18/2019 PT. 7. Protein

110/125

• 0t has been used to determine the proteincontent of milk and meat products• 0t has not been used widely in food system

• 0t is better applied in a purified proteinsystem

• 0t is also better applied in proteins that

have been e6tracted in alkali or denaturingagents such as 9 M urea

B. 2PP

8/18/2019 PT. 7. Protein

111/125

" D:S2D42NT2G5S

8/18/2019 PT. 7. Protein

112/125

#$ Nucleic acid also absorb at :9! nm$:$ &he solution must be clear and colorless$

&urbidity due to particulates in thesolution will increase absorbance falsely

5$ . relatively pure system is re'uired touse this method

. D:S2D42NT2G5S

The >asic Principles o* SomeMethods to Measure Protein 7ontent

8/18/2019 PT. 7. Protein

113/125

III.

8/18/2019 PT. 7. Protein

114/125

A. =&E

8/18/2019 PT. 7. Protein

115/125

at lo p3, basic groups o* protein are (?#charged. These ill uantitati%el& bind a (-#charged d&e.

)hat are these basic groups NH 3 +

CH

CH

CHCH

CH

N

N

H

HC

!

C

CH

C NH+

CH

CH

N

HC

H

C

!

NH

CH

CH

CH

NH

C

NH

NH+

8/18/2019 PT. 7. Protein

116/125

"bsorbance of dye bound by protein% "bsorbance of initial dye "bsorbance

8/18/2019 PT. 7. Protein

117/125

% bsorbance of initial dye bsorbanceof filtrate

2 .

a t " C ! n m

A Protein (K eldahl#

= 1 /! / /" /=

Skim milk

Kadar protein maka intensitas warna filtrat ↓

/ fl i 0i di d i i $

8/18/2019 PT. 7. Protein

118/125

*actors /nfluencing ,ye 0inding determination$

#. 1emperature. Non-proteins.

3. 0uffers systems2. 'rotein uality

8/18/2019 PT. 7. Protein

119/125

8/18/2019 PT. 7. Protein

120/125

• P8:NS:P $ d&e ? protein → kompleks larut

8/18/2019 PT. 7. Protein

121/125

p p• Jang ditera $ absorbansi sen&a a

kompleks &ang larut tersebut• 6leh karena itu $ bila kadar protein ↑ maka

absorbansi uga ↑

• Dengan tabel kon%ersi &ang menun ukkanhubungan antara cat &ang terikat proteindengan kadar protein → kadar proteinsampel dapat diketahui

• Dapat pula dibuat garis regresi &ang &angmenun ukkan hubungan antara cat &angterikat protein dengan kadar protein

76MP28:S6N 6F

8/18/2019 PT. 7. Protein

122/125

#$ S.7P/+ P +P. .&0*N• 1jeldahl > umas methods re'uire little

preparation → sample particle si4e of :!mesh is satisfactory

• *ther methods re'uire fine particles fore6traction of protein from the comple6

food systems

M5T36DS

:$ P 0N%0P/+

8/18/2019 PT. 7. Protein

123/125

• 1jeldahl > umas methods measure

directly the total amount of organic N in thefoods• *ther method measure the various

properties of protein$ ?or e6amples =- &he &rp

5$ S+NS0&020&@

8/18/2019 PT. 7. Protein

124/125

• 1jeldahl8 umas8 and

8/18/2019 PT. 7. Protein

125/125

2K3:8 D28: KU232S2N PROTEIN T58:M2 K2S:3