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Disampaikan oleh
Sri Naruki
Fakultas Teknologi Pertanian UGM
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:5–7%
- 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
• 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 menguap)
2. Penambahan potasium sulfat dapat dila-
kukan untuk menaikkan titik didih asam
sulfat sehingga meningkatkan digesti
Suhu digesti yang ideal : 370 – 410 oC.
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 after
one hour at 370 – 400 oC
a. Procedure of Digestion
• Place sample (accurately weighed) in a
Kjeldahl flask
• Add sulfuric acid and catalyst
UNTUK MENGOKSIDASI MEMERLUKAN H2SO4
1 g Protein + 9,0 g
1 g Lemak + 17,8 g
1 g Karbohidrat + 7,3 g
% 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. 750 nm
A at
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. Relatively simple, can be done in 1 – 1.5 hr
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
R–CH–C–OH + NaOH R–C–C–O–
NH2 NH3+
pada pH netral
H O H
R–C–C–O– + CH2O R–C–COOH
NH3+ (formalin) HOH2C–N–CH2OH
(dimetilol)
H
R–C–COOH H O
HOH2C–N–CH2OH + NaOH R–C–C–ONa
(dimetilol) HOH2C–N–CH2OH
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
- % kasein = 1,63 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 BSA
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
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
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
AKHIR DARI
KULIAH DENGAN
POKOK BAHASAN
PROTEIN
TERIMA KASIH