ANALISIS PROTEIN

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PROTEIN
Protein are an abundant component in
all cells (Nielsen, 2004)
Food protein : very complex; MW
ranging from 5000 to more than a
million Daltons
Function : growth & maintenance of
tissue, formation of essential body
compounds, transportation of nutrients,
etc. (Guthrie, 1982)
 Protein consist of polypeptides,
extremely long chains of many amino
acid units
All protein in all species, regardless
of their function or biological activity,
are built from the same basic set of
20 standard amino acids, which by
themselves HAVE NO intrincsic
biological activity
 General structure of amino acids :
COOH
H2NCH
R
R = side chain or R group
The simples amino acid : glycine R
=H
In all amino acids, except glycine, the
carbon atom has 4 different
substituent groups and is thus
asymetric or chiral carbon
(Lehninger, p. 96)
The Peptide Bond
Peptide bond : how the amino
acids are linked together to make a
protein
H H
H2NCCOOH + H2NCCOOH
R R
H O H
H2N-C-C-N-C-COOH + H2O
R H R
 Proteins are composed of H, C, N, O,
and S (Nielsen, 2004)
Composition of elements in
proteins :
- C : 50 55 %
- O : 20 25 %
- N : 13.4 19.1 %
- H:57%
- S : 0.4 2.5 %
- P, Fe, Cu : trace
 C (karbon) : merupakan penyusun
terbesar
Bila C digunakan sebagai dasar
analisis protein
Analisis protein berdasarkan C
mempunyai beberapa keuntungan :
a. Digesti lebih mudah bila
dibandingkan dengan digesti pada
metoda analisis berdasarkan N
b. Kadar C protein = tinggi sehingga
- mengurangi error
- faktor konversi relatif konstan
 TAPI INGAT : ada beberapa senyawa
lain yang juga mengandung C
karbohidrat dan lipida
Oleh karena itu, C nonprotein
tersebut harus dihilangkan terlebih
dahulu
Pemisahan C nonprotein tersebut
sulit

Oleh karena itu, yang lazim

dilakukan adalah : penentuan kadar
protein berda-sarkan kadar nitrogen
 Nitrogen is the most distinguishing
element present in proteins
N content in various food protein
ranges from 13.4% to 19.1% an
average = 16.0%
Therefore, protein content (%)
= 100/16x % N
= 6.25 x % N
conversion factor/CF (faktor
konversi/FK)
Conversion Factors
N content in various food protein
ranges from 13.4% to 19.1%
conversion factors for various food
are varied :
- Egg or meat CF =
6.25
- Dairy products
6.38
- Wheat
5.70
- Other cereal grains & oilseeds
6.25
ANALYSIS OF PROTEIN
Analysis of protein is very
complicated since some food
components posses similar
physicochemical properties
Nonprotein nitrogen could come from
:
- Free amino acids - Some vitamins
- Small peptide - Alkaloids
- Nucleic acids - Uric acid
- Porphyrin - Urea
Aspartame is a small peptide composed of
Asp, Phe, and methanol
Nucleic acids are polymer formed from linking of
various nucleotides
Struktur vitamin B1 (tiamin) juga mengandung N
Caffeine alkaloid
Chemical structure of uric acid
Chemical structure of urea
 The total organic nitrogen in foods
would represent nitrogen :
- Primarily from protein
- A lesser extent from all organic
nitrogen-containing nonprotein
substances
Therefore, % Protein = CF x % N
is called : crude protein (protein
kasar)
Other major food components,
including carbohydrate and lipid,
may interfere physically with analysis
Importance of Analysis
1. Nutrition labeling
2. Functional property investigation
Proteins of food have unique
functional properties. Example :
gliadin & glutenin in wheat flour for
bread making
3. Biological activity determination,
including enzymes & enzyme
inhibitor. Enzyme activity is
expressed in terms of specific
activity : unit of enzyme/mg of
 Protein analysis is required if you
want to know :
1. Total protein content
2. Content of particular protein in
mixture
3. Protein content during isolation and
purification of protein
4. Nonprotein nitrogen (ex. TMA
content)
5. Amino acid composition (profil asam
amino)
6. Nutritive value of protein (ex. PER,
Protein Content of Selected Foods
Food item % Protein
(wet weight
basis)
CEREALS & PASTA
Rice, brown, raw 7.9
Wheat flour 13.7
Corn flour, yellow 6.9
Spaghetti, dry, enriched 12.8
Cornstarch 0.3
DAIRY PRODUCTS
Milk, whole, fluid 3.3
Cheese, cheddar 24.9
FRUITS AND VEGETABLE
Apple, raw, with skin 0.2
Potato, whole, flesh and skin 2.0
LEGUMES
Soybean, mature seeds, raw 36.5
MEAT, POULTRY. FISH
Egg, raw, whole 12.5
Ham, sliced, regular 17.6
The Basic Principles of Some
Methods to Measure Protein
Content
Determination of nitrogen, peptide
bonds, aromatic amino acids
Based on the dye-binding capacity of
protein
Based on the UV absorptivity of
protein
Based on the light scattering
properties of protein
 The Basic Principles of Some
Methods to Measure Protein
Content
I. Determination of
nitrogen, peptide bonds,
aromatic amino acids

A. Kjeldahl method D. Titrasi

formol
B. Biuret method E. Dumas
method
C. Lowry method F. BCA
A. KJELDAHL
METHOD
 On March 7, 1883, Johann Kjeldahl
presented his method of N analysis
Today, the Kjeldahl method for the
determination of organic nitrogen is
the worldwide standard for the
purpose of calculating the protein
content in both human food and
animal feed
The Kjeldahl method has been
adapted as a standard method of N
analysis in fertilizer, fossil fuel, waste
water, etc.
PRINCIPLE
Proteins & other organic food
components in a sample are digested
with sulfuric acid in the presence of
catalysts
The total organic nitrogen is
converted to ammonium sulfate
The digest is neutralized with alkali
and distilled into a boric acid solution
The borate anions formed are
titrated with standardized acid, which
is converted to N in the sample
 The result of analysis represents the
crude protein content of the food
since N also comes from nonprotein
components
The Kjeldahl method for nitrogen
analysis is composed of three distinct
steps :
1. Digestion (destruksi)
2. Neutralization and Distillation
3. Titration
 IMPROVEMENTS
1. Katalis logam seperti Hg, Cu, dan Se
ditam-bahkan ke asam sulfat untuk
menyempurna-kan oksidasi.
- Yang terbaik : Hg
- Selenium dioksida dan Cu-sulfat (3:1)
juga efektif untuk digesti
- Copper and titanium dioxide also have
been used as a mixed catalyst for
digestion the use of them poses less
safety concern than Hg in the post-
analysis disposal of the waste
 Bila digunakan Hg, selama digesti
akan terbentuk senyawa kompleks
Hg-amonia
Hg harus diendapkan (sebagai HgS)
dengan penambahan Na-tiosulfat
(Na2S2O3) presipitan Na2S2O3
dicampur dengan NaOH yang
digunakan untuk membebas-kan NH3
(saat tahap netralisasi sebelum
distilasi)
Na-tiosulfat juga ditambahkan untuk
membantu membebaskan N dari
 Di lapangan, campuran (NaOH +
Na2S2O3) dikenal dengan istilah
NaOH-thio

Katalis Se (selenium) :
- efek lebih hebat daripada Hg
- Tidak perlu step tambahan (yaitu
penam-bahan Na-tiosulfat /Na2S2O3)
- TAPI : bila jumlah Se berlebihan maka
suhu destruksi/digesti tidak
terkontrol sehingga ada
kemungkinan kehilangan N (karena
2. Penambahan potasium sulfat dapat
dila-kukan untuk menaikkan titik
didih asam sulfat sehingga
meningkatkan digesti
Suhu digesti yang ideal : 370 410
o
C.
TAPI bila K-sulfat berlebihan maka
akan terjadi kehilangan N
3. Sulfide or sodium thiosulfate are
added to the diluted digest to help
release nitrogen from mercury, which
tends to bind to ammonium
4. The ammonia is distilled directly into
a boric acid solution, followed by
titration with standard acid
5. Colorimetry Nesslerization, or ion
chroma- tography to measure
ammonia, is used to determine
nitrogen content after digestion
Beberapa Modifikasi
1. Cara makro Kjeldahl
-. Berat sampel : 1 g
-. Katalis : K2SO4-HgO
-. Distilasi : memapak lempeng Zn
dengan tujuan agar supaya
* tidak terjadi superheating
* tidak terjadi percikan cairan
* tidak terbentuk gelembung gas
yang besar
2. Cara mikro Kjeldahl
- Berat sampel : 10 30 mg
- Katalis : Na2SO4 HgO

3. Cara Gunning
- Berat sampel : 0,7 5,5 g
- Katalis : K2S atau Na2SO4 dan CuSO4
GENERAL PROCEDURE &
REACTIONS
As stated before, the Kjeldahl
method for nitrogen analysis is
composed of three distinct steps.
Sample preparation should be done
prior to these three steps.

1. Sample preparation
2. Digestion (destruksi)
3. Neutralization and Distillation
4. Titration
1. Sample Preparation
Solid food are ground to pass a 20
mesh screen
Samples for analysis should be
homogeneous
No other special preparation are
required
2. Digestion Step
The purpose of digestion : to break
the intricate structure and chemical
bonds that hold a chemical
substances (piece of meat, cup of
flour, or quart of oil) down into
simple chemicals and ionic structures
Specifically, proteins and other forms
of nitrogen are broken down and
converted to ammonia
 To digest the sample, 1 2 g of the
sample are placed on a digestion
tube with 12 15 mL of concentrated
H2SO4. Seven grams of K2SO4 and a
metallic catalyst, usually Cu, are the
added.
The digestion tube is heated to the
boiling temperature of the mixture
Digestion was done to complete
oxidation and conversion of N to
ammonium sulfate
The digestion is usually completed
a. Procedure of
Digestion
Place sample (accurately weighed) in a
Kjeldahl flask
Add sulfuric acid and catalyst
UNTUK MEMERLUKAN H2SO4
MENGOKSIDASI
1 g Protein + 9,0 g
1 g Lemak + 17,8 g
1 g Karbohidrat + 7,3 g
Digest until CLEAR to get complete
breakdown of all organic matter
Digesti diakhiri bila larutan menjadi
jernih dan tak berwarna
b. Reactions During
Digestion
Nonvolatile ammonium sulfate is
formed from nitrogen and sulfuric
acid :
Sulfuric acid
Protein (NH4)2SO4
Heat, catalyst
During digestion :
- protein nitrogen is liberated to form
ammonium ions
- Sulfuric acid oxidized organic matter
& combines with ammonium formed
- C is converted to CO2
(volatile/menguap)
- H is converted to H2O
(volatile/menguap)
Lama digesti bervariasi, tergantung
dari jenis sampel yang dianalisis
- Protein kaya akan asam amino
histidin (His) dan triptofan (Trp) :
digesti lama (perlu katalis lebih
banyak)
c. Reaksi lengkap
HgO + H2SO4 HgSO4 + H2O
HgSO4 Hg2SO4 + SO2 + On

(CHON) + On + H2SO4 CO2 + H2O +

(NH4)2SO4
protein
3. Neutralization &
Distillation Step
Pada tahap ini (NH4)2SO4 dipecah
menjadi NH3 dengan penambahan
NaOH (sampai dica-pai kondisi alkalis)
dan pemanasan (distilasi)
Selama distilasi dapat ditambahkan
lempeng Zn
NH3 yang dibebaskan ditangkap oleh
larutan asam standar berlebihan. Asam
dapat berupa
- Asam borat 4%
- HCl 0,1N
 Ujung alat distilasi harus tercelup
larutan asam
Distilasi diakhiri bila destilat yang
dihasil-kan tidak lagi bereaksi basa.
Caranya ????
Reaksi yang terjadi saat netralisasi
dan distilasi :
(NH4)2SO4 + 2NaOH 2NH3 +
Na2SO4 + 2H2O
Selanjutnya NH3 yang dibebaskan
akan bereaksi dengan larutan
penampung sebagai berikut :
 Bila penampungnya asam borat :
NH3 + H3BO3 (boric acid) NH4 +
H2BO3 (borate ion)

Bila penampungnya HCl :

2NH3 + 2HCl (berlebihan)
2NH4Cl + HCl (sisa)
 Procedure of Netralization &
Distillation
The digest is diluted with water
Alkali-containing sodium thiosulfate
is added to neutralize the sulfuric
acid
The ammonia formed is distilled into
a boric acid solution containing the
indicator methylene blue and methyl
red
4. Titration
a. Bila digunakan penampung
asam borat (METODA TITRASI
LANGSUNG)
-. Borate anion (proportional to the
amount of nitrogen) is titrated with
standardized HCl (anion borat dalam
penampung dititrasi dengan HCl
standar 0,1N )
-. Reaction : H2BO3 + H+ H3BO3
-. Calculation
Moles of HCl = moles of NH3 =
 A reagent blank should be run to
subtract reagent nitrogen from the
sample nitrogen
%N=
N HCl x (corrected acid volume/g of
sample) x (14g N/mol) x 100
Where :
- N HCl = normality of HCl, in
moles/1000ml
- Corrected acid volume =
(ml std acid for sample) (ml std
acid for
 Atau :
% N = (ml HCl sampel ml HCl

blanko) : berat sampel (g) x 1000

x NHCl x 14,008 x 100%

Untuk menghitung kadar protein,

maka % N dikalikan faktor konversi
(CF atau FK)

CF beberapa jenis bahan makanan :
lihat slide sebelumnya
 b. Bila digunakan penampung
HCl (METODA KONVENSIONAL /
BACK TITRATION / TITRASI BALIK)
Bila digunakan penampung HCl,
maka HCl sisa dititrasi dengan NaOH
standar (0,1N)
Reaksi
- Saat distilasi :
2NH3 + 2HCl (berlebihan)
2NH4Cl + HCl (sisa)
- Saat titrasi :
Perhitungan
Pada metoda konvensional, perlu dua
(2) macam larutan standar yaitu HCl
standar dan NaOH standar
meq NH3 hasil distilasi = (meq asam
mula- mula meq asam setelah
distilasi)
meq NH3 = (ml asam x N asam)
(ml NaOH x N NaOH)
Berat N (g) = meq N x 0,014007
Kadar N (%) =
(ml HCl x N HCl) (ml NaOH x N
NaOH)x 1,4
 Atau :
%N =(ml NaOH blanko ml NaOH

sampel) : berat sampel (g) x

1000 x NNaOH x 14,008 x 100%

A factor is used to convert % N to %

crude protein
Most proteins contain 16% N, so the
conversion factor (CF) is 6.25
% Protein = % N x 6.25
PROSEDUR LENGKAP
METODA KJELDAHL
0,7 2.2 g sampel (dalam labu
Kjeldahl)
0,7g HgO (atau 0,65g
logam Hg)
15g K2SO4 (atau Na2SO4
anhidrat)
25ml H2SO4 pekat
DESTRUKSI/DIGESTI (didihkan)
pelan-pelan, tambahkan
sedikit parafin
PENDINGINAN
+ 200 ml aquades dingin
Butiran Zn
25ml larutan Na2S2O3 8%
+ 15g NaOH
PEMASANGAN RANGKAIAN
DISTILASI
PEMASANGAN ERLENMEYER
PENAMPUNG (15ml HCl 0,02N +
6 tetes MR-BCG)
DISTILASI (Diakhiri bila distilat mencapai
> 150ml)
(Cuci ujung kondenser)
PROSEDUR LENGKAP
METODA MIKRO
KJELDAHL
+ 30mg Sampel (dalam labu Kjeldahl)
2g K2SO4; 40mg HgO
2ml H2SO4 pekat
Batu didih
DESTRUKSI/DIGESTI
Bila larutan sudah jernih, tambahkan
aquades dan didihkan lagi + 1,5
jam
PENDINGINAN & PEMINDAHAN KE LABU
PENDINGINAN
8 10ml NaOH-Na2S2O3
DISTILASI
- Tampung distilat dalam
Erlenmeyer berisi :
5ml H3BO3 4% + 4 tetes
indikator (0,2% MR + 0,2% BCG)
- Distilasi diakhiri saat distilat
sudah tidak lagi alkalis
TITRASI
- Distilat dititrasi dengan HCl 0,02N

LAKUKAN PENENTUAN
ALTERNATE
PROCEDURES
In place of distillation & titration with
acid, ammonia or nitrogen can be
quantitated by :
1. NESSLERIZATION :
4NH4OH + 2HgI2 + 4KI + 3KOH
mercuric iodide
NH2Hg2IO + 7KI + 2H2O
ammonium dimercuric iodide
 NH2Hg2IO (red-orange) can be
determined spectrophotometrically at
440nm
This method is rapid & sensitive, but
the ammonium dimercuric iodide is
colloidal and color is not stable
OH
2. NH3 + phenol + hypochlorite
indophenol (blue, 630nm)
3. pH measurement after distillation into
known volume of boric acid
4. Direct measurement of ammonia,
using ion chromatographic method
ADVANTAGES
1. Applicable to all types of foods
2. Inexpensive(if not usingan
automated system)
3. Accurate; an official method for
crude protein content)
4. Has been modified (micro Kjeldahl
method) to measure microgram
quantities of protein)
DISADVANTAGES
1. Measures total organic nitrogen, not
just protein nitrogen
2. Time consuming (at least 2 hr to
complete)
3. Poorer precision than the Biuret
method
4. Corrosive reagent
CONTOH SOAL
Pada analisa kadar protein dengan
metoda Kjeldahl, diperoleh hasil
kadar protein susu bubuk skim
adalah 40%. Penampung distilat
yang digunakan adalah HCl
berlebihan. Bila berat sampel =
0,25g dan titrasi blanko
membutuhkan NaOH 0,1N sebanyak
45ml, hitunglah volume NaOH untuk
titrasi sampel. Faktor konversi N ke
protein skim = 6,38.
B. BIURET METHOD
1. PRINCIPLE
A violet-purple color is produced
when cupric ions are complexes with
> 2 pep-tide bonds, under alkaline
conditions)
The absorbance of the color
produced is read at 540 nm
The color intensity (absorbance) is
proportional to the protein content of
the sample
2. REACTIONS
H O H
HOOCNC C NH2 + CuSO4 + NaOH

R H R
R H H R
2HNCCNCCOOH

HO H
Cu + Na2SO4
+ H2O
H O
 Hasil yang diperoleh dapat
dipengaruhi oleh adanya lipida dan
komponen lain yang dapat
mengubah warna ataupun
memberikan respons yang sama
dengan ikatan peptida.
Contoh gugus yang memberi respons
yang sama dengan ikatan peptida :
CSNH2 CHNH2CH2OH
C(NH)NH2 CHNH2CHOH
CH2NH2 CHOHCH2NH2
CRHNH2
3. PROCEDURE
1. A 5-ml biuret reagent is mixed with
a 1-ml portion of protein solution
(1 to 10mg protein/ml). The reagent
include copper sulfate, NaOH, and K-
Na-tartrate, which is used to
stabilize the cupric ion in the
alkaline solution
2. After the reaction mix is allowed to
stand at room temperature for 15 or
30 min, the absorbance is read at
540 nm against a reagent blank
 Filtration or centrifugation before
reading absorbance is required if the
reaction mixture is not clear
A standard curve of concentration
versus absorbance is constructed
using bovine serum albumin (BSA)

BSA : 0, 20, 40, 60, 80, 100, 120

g/mL
4. ADVANTAGES
Lebih murah daripada metoda
Kjeldahl, cepat (dapat selesai dalam
< 30 menit), paling sederhana
Deviasi warna jarang terjadi
dibanding dengan metoda Lowry,
absorpsi UV, atau turbidimetri
Senyawa yang dapat mengganggu
analisis hanya sedikit sekali
Tidak mendeteksi nitrogen dari
sumbernonprotein
5. DISADVANTAGES
1. Kurang sensitif bila dibandingkan
dengan metoda Lowry; perlu
minimal 2 4 mg protein untuk
analisa
2. Absorbansi dapat berasal dari
pigmen empedu, bila pigemen
tersebut ada di dalam sampel
3. Garam amonium dengan
konsentrasi tinggi dapat
mengganggu reaksi
4. Warna yang dihasilkan bervariasi
dengan perbedaan jenis protein.
Misal : gelatin menghasilkan warna
pinkish-purple
5. Kondisi tak tembus cahaya dapat
terjadi pada larutan final apabila
terdapat lipida atau karbohidrat
dalam jumlah banyak
6. Bukan merupakan metoda yang
absolut : warna harus distandardisasi
dengan protein yang diketahui (misal
BSA) atau dicocokkan dengan
 Hubungan antara absorbansi pada
540 nm pada Biuret method dengan
kadar protein menurut metoda
Kjeldahl
A at 540 nm

% Protein (Kjeldalh)
C. LOWRY METHOD
1. PRINCIPLE
The Lowry method combines the
biuret reaction (see point B) with the
reduction of the Folin-Ciocalteau
phenol reagent (phosphomolybdic-
phosphotungstic acid) by tyrosin &
tryptophan residues in the protein
The bluish color developed is read at
750 nm (high sensitivity for low
protein concentration) or 500 nm
(low sensitivity for high protein
concentration)
Protein reaction with cupric ion under
alkaline condition
2a. PROSEDUR
Terdapat 2 macam reagen Lowry,
yaitu
- Lowry A (mengandung fosfotungstat-
fosfo molibdat 1 : 1)
- Lowry B (mengandung Na-karbonat
2% dalam NaOH 0,1N serta Cu-sulfat
dan Na-K-tartrat 2%)
 Cara :
- 1 mL larutan protein sampel + 5mL
reagen Lowry B, gojog dan biarkan
10 menit
- Kemudian tambahkan 0,5 mL reagen
Lowry A dan biarkan 20 menit
- Baca nilai absorbansi pada 600 nm
2b. PROCEDURE
1. Proteins to be analyzed are diluted
to an appropriate range (20 100
g)
2. K Na Tartrate-Na2CO3 solution is
added after cooling and incubated
at room temperature for 10 min
3. CuSO4-K Na Tartrate-NaOH solution
is added after cooling and incubated
at room temperature for 10 min
4. Freshly prepared Folin reagent is
added,
then the reaction mixture is
mixedand incubated at 50oC for 10
min
5. Absorbance is read at 650 nm
6. A standard curve of bovine serum
albumin (BSA) is carefully
constructed for estimating protein
concentration of the unknown
 Cu++ in alkaline solution to form complexity with
protein.
Cu++ catalyses oxidation of phenol group of tyrosine
with phosphomolybdic-phosphotungstic acid.
at 750 nm
A

g of protein (Kjeldahl)
3. ADVANTAGES
1. Very sensitive :
a. 50 100 times more sensitive than
biuret method
b. 10 20 times more sensitive than
280nm UV absorption method
c. Similar sensitivity as Nesslerization;
however, more convenient than
Nesslerization
2. Less affected by turbidity of the
sample
3. More specific than most other
methods
4. DISADVANTAGES
1. Color varies with different proteins to a
greater extent than biuret method
2. Color is not strictly proportional to
protein concentration
3. The reaction is interfered with to
varying degrees by sucrose, lipids,
phosphate buffers, monosaccharides,
and hexoamines
4. High concentration of reducing sugars,
ammonium sulfate, and sulfhydryl
compounds interfere with the reaction
D. TITRASI FORMOL
1. PRINSIP
Larutan protein dinetralkan dengan
basa (NaOH), kemudian ditambah
formalin sehingga terbentuk dimetilol
Dengan terbentuknya dimetilol maka
gugus amino protein sudah terikat
dan tidak mempengaruhi reaksi
antara asam (gugus karboksil)
dengan basa NaOH sehingga akhir
titrasi dapat diakhiri dengan tepat
 Indikator yang dipakai adalah PP
Akhir titrasi ditandai saat terjadinya
perubahan warna menjadi merah
muda yang tidak hilang dalam 30
detik
Titrasi formol baik untuk digunakan
untuk evaluasi proses terjadinya
pemecahan protein (misal : pada
fermentasi protein pada tempe,
kecap, tauco, dzsb.)
Proses hidrolisis protein ditandai
dengan meningkatnya titrasi formol
2. REAKSI PADA TITRASI
FORMOL
O H O
RCHCOH + NaOH RCCO
NH2 NH3+
pada pH netral
H O H
RCCO + CH2O RCCOOH
NH3+ (formalin) HOH2CN
CH2OH

(dimetilol)
H
RCCOOH H
O
3. PROSEDUR
10 ml larutan protein sampel + 20 ml
aqu-ades + 0,4 ml larutan K-oksalat
jenuh (K-oksalat : air = 1 : 3) dan 1 ml
indikator PP 1%. Diamkan selama 2
menit
Titrasi larutan tersebut dengan 0,1N
NaOH sampai terbentuk warna pink
atau warna standar (10 ml susu + 10
ml aquades + 0,4 ml K-oksalat jenuh +
1 tetes indikator rosanilin-khlorida
0,01%)
 Setelah warna tercapai, tambahkan 2
ml formaldehid 40% dan titrasilah
kembali dengan larutan NaOH
sampai warna seperti warna standar
tercapai lagi. Catatlah nilai titrasi
kedua ini.
Dibuat titrasi blanko yang terdiri
dari : 20 ml aquades + 0,4 ml larutan
K-oksalat jenuh + 1 ml indikator PP +
2 ml formal-dehid, dan titrasilah
dengan larutan NaOH
Titrasi formol = titrasi terkoreksi
= titrasi kedua titrasi blanko
 Bila nilai titrasi formol akan
digunakan untuk menentukan kadar
protein, maka harus dibuat
percobaan serupa dengan
menggunakan larutan yang telah
diketahui kadar proteinnya (misalnya
dengan metoda Kjeldahl)
Selanjutnya ditentukan hubungan
antara titrasi formol dengan %
protein.
Misal :
- % protein susu = 1,83 x ml titrasi
formol
E. DUMAS
(NITROGEN
COMBUSTION)
METHOD
1. PRINCIPLE
Sample are combusted at high tempera-
ture (700 1.000oC)
The nitrogen released is quantitated by
gas chromatography using thermal
conductivity detector (TCD)
The nitrogen determined is converted to
protein content in the sample
2. PROCEDURE &
APLICATION
PROCEDURE
Sample (100 500 mg) are weighed into
a tin capsule and introduced to a combust-
on reactor in automated equipment
The nitrogen released is measured by a
built-in gas chromatograph
APLICATION
It is an alternative to the Kjeldahl method
It is suitable for all type of foods
3. ADVANTAGES &
DISADVANTAGES
ADVANTAGES
Requires no hazardous chemicals
Can be accomplished in 3 minutes
Recent automated instrument can analyze
up to 150 samples without attention
DISADVANTAGES
Expensive equipment is required
Measures total organic nitrogen, not just
protein nitrogen
F. BICINCHONINIC
ACID (BCA) METHOD
1. PRINSIP
Protein mampu mereduksi ion kupri
(Cu2+) menjadi ion kupro dalam
suasana alkalis
Dengan reagen BCA (berwarna
apple-greenish), ion kupro tersebut
membentuk kompleks berwarna
purplish, yang intensitasnya dapat
ditera pada 562 nm
Intensitas warna purplish tersebut
propor-sional dengan kadar protein
2. PROSEDUR
Mix (one step) the protein solution
with the BCA reagent, which contain
BCA sodium salt, Na-carbonate,
NaOH, and Cu-sulfate pH 11.25
Incubate at room temperature for 2
hr, or 60oC for 30 min. A higher
temperature gives a greater color
respon
Read the solution at 562 nm against
a reagent blank
Construct a standard curve using
3. APPLICATION
BCA method had been used in
protein isolation and purification
The suitability of BCA method for
measuring protein in complex food
has not been reported
4. ADVANTAGES
1. Sensitivity of the micro BCA method
(0.5 10 g) is better than Lowry
method
2. One-step mixing is easier than in
the Lowry method
3. The reagent is more stable than for
the Lowry method
4. Nonionic detergent and buffer salts
do not interfere with the reaction
5. DISADVANTAGES
1. Color is not stable with time. The
analyst needs to carefully control
the time for reading absorbance
2. Any compound capable of reducing
Cu2+ to Cu+ will lead to color
formation
3. Reducing sugar interfere to a
greater extent than in Lowry method
4. Color variation among proteins are
similar to those in the Lowry method
The Basic Principles of Some
Methods to Measure Protein Content

II. Based on the UV

absorptivity of protein
ULTRAVIOLET (UV)
280 nm METHOD
1. PRINCIPLE
Protein show strong absorption at
280 nm, primarily due to tyrosine
(Tyr) and tryptophan (Trp) residues in
the protein
Because the content of Tyr & Trp in
protein from each food source is
fairly constant, the A280 could be used
to estimate the concentration of
protein
2. PROCEDURE
Protein are solubilized in buffer or
alkali
Absorbance of protein solution is
read at 280 nm against a reagent
blank
Protein concentration is calculated
according Beers law : A = abc
A = absorbance, a = konstanta (molar
absorp- tivity for individual protein), b
= cuvette path length, c =
concentration
3. APPLICATION
It has been used to determine the protein
content of milk and meat products
It has not been used widely in food system
It is better applied in a purified protein
system
It is also better applied in proteins that
have been extracted in alkali or denaturing
agents such as 8M urea
4. ADVANTAGES
1. Rapid and relatively sensitive (at 280 nm,
+ 100g protein is required, several times
more sensitive than the Biuret method)
2. Nondestructive : sample can be used for
other analysis after protein determination
3. No interference from ammonium sulfate
and other buffer salts
4. DISADVANTAGES
1. Nucleic acid also absorb at 280 nm.
2. The solution must be clear and colorless.
Turbidity due to particulates in the
solution will increase absorbance falsely
3. A relatively pure system is required to
use this method
The Basic Principles of Some
Methods to Measure Protein Content

III. Based on the dye-binding

capacity of protein
A. Dye : acid orange-12
B. Dye : coamassive brillian blue
G-250
A. DYE BINDING METHOD
with acid orange-12 as a
dye
Principle:
at low pH, basic groups of protein are (+)
charged. These will quantitatively bind a (-)
charged dye.
What are these
NH3
+ basic groups?
CH2 H
+
CH2 CH2 N C NH2
Lysine
CH2 CH2 NH 2
CH2 O CH2 Arginine
H
CH N C CH
N C C N
H H
O CH2
C NH+

HC CH Histidine
N
H
Acid Orange 12: SO3
-

HO
N=N

Procedure:
1. Mix protein, dye, buffer pH = 2. protein +
dye : membentuk komplex tidak larut
2. Filter or centrifuge.
3. Measure optical density (OD) or absorbancy
of filtrate (filtrat mengandung dye sisa yang tak
bereaksi dengan protein)
Absorbance of dye bound by protein
= Absorbance of initial dye Absorbance
of filtrate
Kadar protein maka intensitas warna filtrat
A. at 470 nm

Skim milk

6 8 10 12 14 16

% Protein (Kjeldahl)
Factors Influencing Dye Binding determination:
1. Temperature
2. Non-proteins.
3. Buffers systems
4. Protein quality
 B. DYE BINDING
METHOD
with coamassie brillian
blue G-250 as a dye
 PRINSIP : dye + protein kompleks larut
Yang ditera : absorbansi senyawa
kompleks yang larut tersebut
Oleh karena itu : bila kadar protein maka
absorbansi juga
Dengan tabel konversi yang menunjukkan
hubungan antara cat yang terikat protein
dengan kadar protein kadar protein
sampel dapat diketahui
Dapat pula dibuat garis regresi yang yang
menunjukkan hubungan antara cat yang
terikat protein dengan kadar protein
COMPARISON OF
METHODS
1. SAMPLE PREPARATION
Kjeldahl & Dumas methods require little
preparation sample particle size of 20
mesh is satisfactory
Other methods require fine particles for
extraction of protein from the complex
food systems
2. PRINCIPLE
Kjeldahl & Dumas methods measure
directly the total amount of organic N in
the foods
Other method measure the various
properties of protein. For examples :
- The Biuret method measures peptide
bonds
- The Lowry methos measures a
combination of peptide bonds and the
amino acids Tyr & Trp
3. SENSITIVITY
Kjeldahl, Dumas, and Biuret method are
less sensitive than Lowry, BCA, or UV
method

4. SPEED
Speed of determination in spectrophoto-
metric method and the Dumas method are
faster than with the Kjeldahl method