Analysis of Protein

ANALISIS PROTEIN Widiastuti Setyaningsih
TPHP-FTP-UGM
Widiastuti Setyaningsih widisety.com
PROTEIN CONTENT OF SELECTED FOODS

•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, transpor-
tation of nutrients, antibody and protective tissue,
contractive tissue, bio-catalysator (enzyme), etc.
(Guthrie, 1982)
THE IMPORTANCE OF ANALYSIS

1. Nutrition labeling and pricing
2. Functional property investigation
3. Biological activity determination
1.Nutrition labeling: Protein content is one of the mandatory items in the
standard format of the “Nutrition Facts” label.
2.Pricing: The cost of certain products is based on the protein content (e.g.,
cereal grains; milk for making certain dairy products as cheese).
3.Functional property investigation: Proteins in various types of food have
unique functional properties: for example, casein protein in milk for
coagulation into cheese products, and egg albumin for foaming. Proteins of
food have unique functional properties. Example : gliadin & glutenin in wheat
flour for bread making
4.Biological activity determination: Enzymes are examples of proteins that can
affect the biological activity of food. Examples: proteolytic enzymes affect the
tenderization of meats and pectinase enzymes affect the ripening of fruits.
Biological activity determination, including enzymes & enzyme inhibitor.
Enzyme activity is expressed in terms of specific activity : unit of enzyme/mg
of protein
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 (eg. TMA content)
5. Amino acid composition (profil asam amino)
6. Nutritive value of protein (eg. PER, BV)
7. Activity (of enzyme)
COMPOSITION OF ELEMENTS IN PROTEINS

Proteins are composed of H, C, N, O, and S (Nielsen, 2004)
C : 50 – 55 % Penyusun terbesar
O : 20 – 25 %
N : 13.4 – 19.1 % Penentuan kadar protein
H:5–7%
S : 0.4 – 2.5 %
P, Fe, Cu : trace
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
-Ammonium ions - Etc
PROTEIN CONTENT
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 (%) = 100/16x % N
= 6.25 x % N
conversion factor or CF (=faktor konversi atau FK)
•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)
CONVERSION FACTORS (CF = FK)

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
-Almonds 5.18
-Peanuts & Brazil nuts 5.46
-Other tree nuts & coconut 5.30
WHERE TO FIND THE CONVERSION FACTORS?

WHERE TO FIND THE CONVERSION FACTORS?

THE BASIC PRINCIPLES OF SOME METHODS TO

MEASURE PROTEIN CONTENT
I. Determination of nitrogen, peptide bonds, aromatic amino acids
II. A.Based
Kjeldahl
on themethod
UV absorptivity of protein
B. Biuret method
III. Based on the dye-binding capacity of protein
C. Lowry method
D. Titrasi formol
E. Dumas method
F. BCA method
KJELDAHL METHOD
PRINCIPLE (MICRO KJELDAHL METHOD)

• 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 and alkalinized 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
Link for video: http://bit.ly/22m3dN2

The Kjeldahl method may be broken down into three main steps:
Digestion - the decomposition of nitrogen in organic samples utilizing a
concentrated acid solution. This is accomplished by boiling a homogeneous
sample in concentrated sulfuric acid. The end result is an ammonium (NH4 )-
sulfate solution.
Distillation - adding excess alkali to the acid digestion mixture to convert
NH4 + to NH3, followed by boiling and condensation of the NH3 gas in a
receiving solution (acid solution).
Titration - to quantify the amount of ammonia in the receiving solution.
The amount of nitrogen in a sample can be calculated from the quantified

amount of ammonia ions in the receiving solution.
•Methoda pada slide didepan adalah methoda Kjeldahl Mikro
•Pada methoda Kjeldahl Makro : larutan penampung distilasi adalah
larutan HCl standar . HCl yang tidak bereaksi dengan ammonia is
kemudian dititrasi dengan larutan alkali standar .
Persamaan reaksi :
• Kjeldahl mikro : NH3 + as. borat  NH3 –borat
NH3 –borat dititrasi dengan larutan HCl standar
Perhitungan  miligrek NH3 = miligrek HCl titran
• Kjeldahl makro : NH3 + HCl standar (berlebih, terukur)

sisa HCl dititrasi dengan larutan NaOH standar
Perhitungan  miligrek NH3 = miligrek HCl – miligrek NaOH
PRINCIPLE Link for video: http://bit.ly/1QZ82Vh

ADVANTAGES
1. Applicable to all types of foods
2. Inexpensive (unless by 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
P E P T I D A ; PROTEIN ADALAH POLIPEPTIDA
H H H H
NH3+ C CO NH C CO NH C CO NH C COO-
Ikatan peptida
R1 R2 R2 R2
n
N-terminal Second A.A C-terminal
BIURET METHOD
A violet-purple color is produced when
cupric ions are complexes with > 2
peptide bonds (under alkaline conditions)
The color intensity (at 540 nm) is

proportional to the protein content of
the sample
Link for video: http://bit.ly/1KqojQT
•Berlaku untuk semua polipeptida, protein dan proteida,
karena semua mengandung ikatan peptida, dapat
dikembangkan menjadi metode kolorimetri yang sifatnya
kuantitatif.
•Untuk melihat adanya ikatan peptida, reaksi biuret telah
digunakan untuk mengikuti proses hidrolisis dalam pembuatan
hidrolisat protein dalam industri makanan.
•Hidrolisat protein hewani misalnya boillon yang dibuat pabrik
maggi, hidrolisat kedelai yang dibuat di Jepang adalah contoh
dari hidrolisat protein nabati.
• Saus ikan, petis daging/ikan/udang, saus kedelai (kecap dan
tauco) merupakan produk hidrolisat proten = peptida
BIURET METHOD
Hasil yang diperoleh
dapat dipengaruhi oleh
adanya lipida dan
komponen lain yang
dapat mengubah warna
ataupun memberikan
respons yang sama
dengan ikatan peptida
Prinsip kerja penentuan kadar protein dengan metode biuret adalah menganalisa adanya ikatan
peptida dengan cara menambahkan reagen biuret ke dalam sampel yang kemudian diukur
absorbansinya menggunakan spektrofotometer, reaksinya adalah sebagai berikut:
Pada tes biuret ini penambahan NaOH 10% pada protein menyebabkan terjadinya hidroisis ikatan
peptida dari polimer protein. Hidrolisis ini menghasilkan monomer-monomer asam amino dan ada
sebagian gugus asam amino yang berubah menjadi amonia. Akibatnya hidrolisis itu jumlah gugus
asam amino berkurang dan pengukuran serapan cahaya oleh ikatan kompleks yang berwarna ungu
dapat terjadi; oleh sebab itu protein bereaksi dengan tembaga dalam ingkungan alkali.
Pada dasarnya suatu peptida adalah asi-asam amino karena gugus –COOH dan –NH2 membentuk
ikatan peptida. Peptida didapatkan dari hidrolisis protein yang tidak sempurna. Apabila peptida
yang dihasilkan dihidrolisis lebih anjut akn dihasilkan asam-asam amino.
Sifat peptida ditentukan oleh gugus –COOH, -NH2 dan gugus R. Sifat asam dan basa pada peptida
ditentukan oleh gugus –COOH dan –NH2, namun pada rantai panjang gugus –COOH dan –NH2
yang terletak diujung rantai tidak lagi berpengaruh. Suatu peptida juga mempunyai titik isolistrik
seperti pada asam amino. Reaksi biuret merupakan reaksi warna untuk peptida dan protein.
Filtration or centrifugation before reading absorbance is required if the reaction mixture is not
clear
PROCEDURE
1. A 5-ml biuret reagent (copper sulfate, NaOH, and K-Na-
tartrate) is mixed with a 1-ml of protein solution
2. After the reaction mix is allowed to stand at room
temperature for 15 or 30 min
3. The absorbance is read at 540 nm
PROCEDURE
A standard curve of concentration versus absorbance
is constructed using bovine serum albumin (BSA)
BSA : 0, 20, 40, 60, 80, 100, 120 g/mL

• Prinsip dari spektrofotometri adalah suatu metoda analisa yang didasar -
kan pada pengukuran serapan sinar monokromatis oleh suatu kolom
larutan berwarna pada panjang gelombang spesifik dengan menggunakan
monokromator prisma atau kisi difraksi dengan detektor fototube.
• Metode ini dapat digunakan untuk sampel yang berupa arutan berwarna
atau tidak berwarna, karena pada umumnya suatu alat spektrofotometri
yang dilengkapi sumber cahaya untuk mengukur spektrum dan panjang
gelombang pada arutan tertentu.
• Jumah sinar yang diserap atau diteruskan oleh suatu larutan merupakan
suatu fungsi eksponensial dari konsentrasi larutan dan pada lebar larutan
yang dilalui sinar.
ADVANTAGES
• Lebih murah daripada metoda Kjeldahl
• Sederhana, cepat (dapat selesai dalam < 30 menit)
• Deviasi warna jarang terjadi dibanding dengan metoda Lowry,
absorpsi UV, atau turbidimetri
• Senyawa yang dapat mengganggu analisis hanya sedikit sekali
• Tidak mendeteksi nitrogen dari sumber nonprotein
DISADVANTAGES
1. Kurang dapat menera protein dengan konsentrasi rendah bila
dibandingkan dengan metoda Lowry; perlu minimal 2 – 4 mg
protein untuk analisa
2. Absorbansi dapat berasal dari pigmen empedu, bila pigmen
tersebut ada di dalam sampel
3. Garam amonium dengan konsentrasi tinggi dapat mengganggu
reaksi
DISADVANTAGES
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
metoda Kjeldahl
LOWRY METHOD
•The Lowry method combines the biuret reaction 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 (for low protein concentration)
or 500 nm (for high protein concentration)
PEREAKSI
Terdapat 2 macam reagen Lowry, yaitu
-Lowry A (mengandung fosfotungstat-fosfomolibdat 1 : 1)
-Lowry B (mengandung Na-karbonat 2% dalam NaOH 0,1N serta Cu-sulfat
dan Na-K-tartrat 2%)
PROSEDUR
• 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
• A standard curve of bovine serum albumin (BSA) is carefully
constructed for estimating protein concentration of the unknown
ADVANTAGES
• Very sensitive :
50 – 100 times more sensitive than biuret method
10 – 20 times more sensitive than 280nm UV absorption method
• Less affected by turbidity of the sample
• More specific than most other methods
• Relatively simple, can be done in 1 – 1.5 hr
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
TITRASI (SØRENSEN) FORMOL

A method of titrating the amino groups of amino acids, by adding
formaldehyde to the neutral solution.
The formaldehyde reacts with the NH3+ group, liberating an equivalent

quantity of H+, which may then be estimated by titration with NaOH.
REAKSI PADA TITRASI FORMOL

PROSEDUR
• 10 ml sampel + 20 ml aquades + 0,4 ml larutan K-oksalat jenuh
dan 1 ml indikator PP 1%. Diamkan selama 2 menit
• Titrasi larutan tersebut dengan 0,1N NaOH sampai terbentuk warna
pink atau warna standar
• 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
PROSEDUR
• 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
% kasein = 1,63 x ml titrasi formol
DUMAS (NITROGEN COMBUSTION) METHOD

Sample (100 – 500 mg) are combusted at high temperature (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

SCHEME
DUMAS SYSTEM Link for video: http://bit.ly/1UILGtz

ADVANTAGES
• Requires no hazardous chemicals
• Can be accomplished in 3 minutes
• Recent automated instrument can analyse up to 150 samples
without attention
DISADVANTAGES
• Expensive equipment is required
• Measures total organic nitrogen, not just protein nitrogen
F. BICINCHONINIC ACID (BCA) METHOD

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 proporsional dengan kadar protein
PROCEDURE
•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 response
•Read the solution at 562 nm against a reagent blank
•Construct a standard curve using BSA
•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
ADVANTAGES
• LOD of the micro BCA method (0.5 – 10 g) is better than Lowry
method
• One-step mixing is easier than in the Lowry method
• The reagent is more stable than for the Lowry method
• Nonionic detergent and buffer salts do not interfere with the
reaction
DISADVANTAGES
• Color is not stable with time. The analyst needs to carefully control the
time for reading absorbance
• Any compound capable of reducing Cu2+ to Cu+ will lead to color
formation
• Reducing sugar interfere to a greater extent than in Lowry method
• Color variation among proteins are similar to those in the Lowry method
II. BASED ON THE UV ABSORPTIVITY OF PROTEIN

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
PROCEDURE
Absorbance of protein solution is read at 280 nm against a reagent blank
Protein concentration is calculated according Beer’s law : A = abc
A = absorbance
a = konstanta (molar absorptivity for individual protein),
b = cuvette path length
c = concentration
Bisa juga memakai standar BSA
ADVANTAGES
• Rapid
• Low LOD and LOQ
• Nondestructive
• No interference from ammonium sulfate and other buffer salts
DISADVANTAGES
• Other substances also absorb at 280 nm.
• The solution must be clear and colorless. Turbidity due to particulates in
the solution will increase absorbance falsely
• A relatively pure system is required to use this method
III. BASED ON THE DYE-BINDING CAPACITY OF PROTEIN

A. Dye : acid orange-12
B. Dye : coamassive brillian blue G-250
DYE BINDING METHOD

WITH ACID ORANGE-12 AS A DYE
+
Principle: NH3
CH2 H
+
CH2 CH2 N C NH2
at low pH, basic groups of protein are CH2
Lysine
CH2 NH2
(+) charged. These will CH2

CH
H
N
O
C
CH2
CH
Arginine
quantitatively bind a (-) charged dye. N

H
C C
CH2
N
H
O
C NH+
What are these basic groups? HC CH Histidine
N
H
DYE BINDING METHOD HO

SO3
-
WITH ACID ORANGE-12 AS A DYE N=N
Procedure:
1. Mix protein, dye, buffer pH = 2.  protein + dye : membentuk komplex tidak larut
2. Filter or centrifuge.
3. Measure the 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 

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
END OF LECTURE

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ANALISIS PROTEIN Widiastuti Setyaningsih

PROTEIN CONTENT OF SELECTED FOODS

THE IMPORTANCE OF ANALYSIS

PROTEIN ANALYSIS IS REQUIRED IF YOU WANT TO

COMPOSITION OF ELEMENTS IN PROTEINS

Nonprotein nitrogen could come from :

CONVERSION FACTORS (CF = FK)

WHERE TO FIND THE CONVERSION FACTORS?

WHERE TO FIND THE CONVERSION FACTORS?

THE BASIC PRINCIPLES OF SOME METHODS TO

PRINCIPLE (MICRO KJELDAHL METHOD)

Link for video: http://bit.ly/22m3dN2

The amount of nitrogen in a sample can be calculated from the quantified

• Kjeldahl makro : NH3 + HCl standar (berlebih, terukur)

PRINCIPLE Link for video: http://bit.ly/1QZ82Vh

The color intensity (at 540 nm) is

BSA : 0, 20, 40, 60, 80, 100, 120 g/mL

TITRASI (SØRENSEN) FORMOL

The formaldehyde reacts with the NH3+ group, liberating an equivalent

REAKSI PADA TITRASI FORMOL

DUMAS (NITROGEN COMBUSTION) METHOD

The nitrogen released is quantitated by gas chromatography using thermal

The nitrogen determined is converted to protein content in the sample

DUMAS SYSTEM Link for video: http://bit.ly/1UILGtz

F. BICINCHONINIC ACID (BCA) METHOD

II. BASED ON THE UV ABSORPTIVITY OF PROTEIN

III. BASED ON THE DYE-BINDING CAPACITY OF PROTEIN

DYE BINDING METHOD

(+) charged. These will CH2

quantitatively bind a (-) charged dye. N

DYE BINDING METHOD HO

WITH ACID ORANGE-12 AS A DYE N=N

Absorbance of dye bound by protein = Absorbance of initial dye – Absorbance of filtrate

Kadar protein  maka intensitas warna filtrat 

DYE BINDING METHOD

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