protein analysis - Farmasi Carbon 2012

PROTEIN ANALYSIS
Abdul Rohman
Laboratorium Kimia Analisis, Bagian Kimia Farmasi, Fakultas Farmasi
UGM
Protein
O
O
H2N
CH
OH +
C
H2N
O
CH
OH + .........
C
R
asam amino
R
asam amino
H2N
CH
C
O
H
N
R
CH
R
ikatan peptida
C
O
H
N
CH
n R
ikatan peptida
C
OH
Protein Content of Some Foods
Milk (dry)
22-25
Rice
Milk (skim)
3.2
Wheat flour
Egg (dry)
35
Walnuts
15-21
Beef (dry)
81-90
Potato
10-13
7.5-9
9.8-13.5
PROTEIN ANALYSIS

Protein analysis is required to know:
1.
2.
3.
4.
5.
6.
total protein
amino acid composition
amount of a particular protein in a mixture
protein content during isolation and purification
Nonprotein nitrogen
Nutritional value of a protein
Methods for Protein Analysis
•
•
•
•
Kjeldahl (Volumetri)
Formol (Volumetri)
Spektroskopi (Biuret dan Lowry)
Chromatography
Kjeldahl method
• Crude protein content
• Johan Kjeldahl (1883) developed the basic process
• Principle: total organic N released from sample and absorbed
by acid
6
Protein analysis in foods has been mostly done
by determining Nitrogen Content
• Nitrogen is: largely unique to protein – only
MAJOR constituent of foods containing N.
• Other nitrogenous compounds (NPN):
Chlorophyll, nucleic acids, some vitamins,
lecithins, urea, amino sugars, alkaloids,
ammonium ions, etc.
• On the average, food proteins contain 16%
nitrogen. 100% divided by 16% = 6.25. Therefore
multiply N content by 6.25 to get protein
content.
Protein analysis in foods has been mostly
done by: Determining Nitrogen content
• The most common and well accepted method
for determining nitrogen in food is the Kjeldahl
method….(Kel-Dall)
• Labconco is one of the industry leaders in
Kjeldahl equipment:
• http://www.labconco.com/pdf/kjeldahl/index.shtml
• Click on “Kjeldahl” and download the brochures.
(A Guide to Kjeldahl Nitrogen Determination Methods and Apparatus)
Kjeldahl method
3 Stage Process:
• 1. Digestion with acid + catalysis
• 2. Distillation with steam and alkali
• 3. Titration with acid and indicators.
Kjeldahl method
• Based on conversion of protein nitrogen to
ammonia (NH3). At the same time, carbon and
hydrogen are oxidized to carbon dioxide and
water.
• In the presence of sulfuric acid (H2SO4), the
ammonia picks up a hydrogen forming
ammonium sulfate (NH4)2SO4.
• Then concentrated sodium hydroxide (NaOH) is
added to the solution of ammonium sulfate +
sulfuric acid. This raises the pH transforming
ammonium (NH4+) into ammonia.
Kjeldahl method
• When the sample containing ammonia, sodium
hydroxide and sodium sulfate is heated, the ammonia
is driven off as a gas.
• We can condense the ammonia gas and introduce it
into a fluid that contains boric acid and pH indicators.
• Ammonia reacts with boric acid to form ammonium
borate. This is a base.
• Titrate ammonium borate to an endpoint with
hydrochloric acid (we must know the normality of the
hydrochloric acid used).
Total organic nitrogen - Kjeldahl
method
• Calculation
%Protein = %N  conversion factor
Conversion factor: generally 6.25
• most protein: 16% N
egg or meat
milk
wheat
soybean
rice
Conversion factor
6.25
6.38
5.33
5.52
5.17
13
Kjeldahl Apparatus
14
Kjeldahl method
• Advantages:
• applicable to any foods
• simple, inexpensive
• accurate, official method for crude protein content
• Disadvantages:
• measuring total N not just protein N
• time consuming
• corrosive reagents
16
ALTERNATES TO DISTILLATION AND
TITRATION
1. NESSLERIZATION = reaction of ammonia with
mercuric iodide to produce ammonium
dimercuric iodide which can be read in a SPEC
(visible @ 440nm)
2. Reaction with phenol and hypochlorite to form
indophenol which can be read in a SPEC (visible
@ 630nm)
Titrasi Formol
• Pada titrasi formol digunakan formaldehid untuk menutup
gugus amin dan membentuk metilol.
• Metode ini digunakan untuk penetapan kadar protein dalam
susu secara cepat.
• Oleh karena protein mempunyai gugus karboksilat dan gugus
amina, maka protein bersifat netral. Bila gugus –NH2
dinonaktifkan oleh formaldehid menjadi bentuk dimetilol,
maka gugus karboksilat akan bersifat asam yang selanjutnya
dapat dititrasi secara alkalimetri dengan larutan baku NaOH
Reaksi antara protein dengan formaldehid membentuk
dimetilol. Gugus karboksilat bebas selanjutnya dititrasi dengan
NaOH
O
H2N
CH
C
O
H
N
R
CH
CH
H
N
N-dimetilol
CH
n R
O
O
C
H
N
CH
HOH2C
R
2 HCOH
R
HOH2C
N
C
O
R
C
C
OH
NaOH
O
H
N
CH
n R
C
OH
Spectroscopic-based techniques
• UV spectroscopy
• Visible spectroscopy
•
•
•
•
•
•
BIURET
FOLIN
LOWRY
NINHYDRIN
DYE BINDING METHOD
BICINCHONIC ACID (BCA) METHOD
UV Absorption (280 nm)
•
Most proteins exhibit strong UV light
absorption at 280 nm because they contain
“chromophoric” side chains such as tyrosine,
tryptophan, and phenylalanine.
•
The concentration of protein in a non-turbid
solution is proportional to the absorbance
UV ABSORPTION (280 nm)
Advantages
- rapid and sensitive
-Nondestructive
-Disadvantages
- nucleic and phenolic acids also absorb at 280 nm
- amounts of Trp and Tyr vary with protein types
- turbidity (cloudiness in solution) is a problem
Applications of method? Not widely accepted for
general food analysis – more useful for research
purposes by monitoring the extraction or
separation of proteins
Biuret Method
•
•
•
Cupric ions react with peptide bonds under alkaline
conditions
(copper sulfate + K-Na-tartrate + alkali)
Measure color in SPECTOPHOTOMETER at 540 nm
O
O
O
H N
H
R
Cu
O
OH-
H N
2+
H N
H
R
O
H N
Cu
2+
N H
H
R
O
N H
Purple biuret complex
BIURET METHOD
Advantages:
- Cheaper and faster than Kjeldahl
- Less problem with color deviations
- Few substances interfere
- Does not measure non protein nitrogen (NPN)
Disadvantages:
-not sensitive: to 2-4 mg level
-color depends on protein
-- PROTEIN MUST BE SOLUBLE
Folin-Ciocalteu
• Used to determine reducing compounds
• Reduction of phosphomolybdic-phosphotungstic acid by
reducing groups in a sample.
• Once reduction has occurred, the solution is made alkaline and
a blue color is formed.
• Can be used directly for proteins, pure sugar solutions, or
phenolic compounds.
LOWRY METHOD
•
The Lowry method is a colorimetric method
based on the formation of a blue color
formed
•
Tyrosine and/or tryptophan in a protein
reduces a phosphomolybdicphosphotungstic reagent (Folin-Ciocalteu
reagent) in the presence of K-Na-tartrate in
alkali (Biuret reagent)
•
Absorbance values are determined on a
spectrophotometer at 750 nm.
As little as 0.2 mg protein in a sample can be
determined.
•
LOWRY METHOD- Advantages
•
•
•
•
This is the most sensitive spectrophotometric
method available for determining total
protein. It is 10 to 20 times more sensitive
than UV absorption at 280 nm (next topic)
This method is more specific
The Lowry method is relatively rapid,
requiring 1-2 hours for analysis
Widely used in biomedical field
LOWRY METHOD- Disadvantages
This method requires careful standardization (making a
good standard curve) because
a. The amount of color varies with different proteins.
b. The color is not strictly proportional to the
concentration
c. Recent evidence suggests that sucrose, lipids, some
buffers, monosaccharides and hexosamines react to
varying degrees with the reagents in the Lowry test
d. High concentrations of ammonium sulfate, sulfhydryl
compounds, and phosphate can interfere
Ninhydrin
•
•
Primary amino groups on the end of proteins,
peptides, and free amino acids will react with
ninhydrin.
This reaction forms a strongly colored purple
solution referred to as Ruheman's purple.
Read at 570 nm.
Ninhydrin
Advantages are
•Faster and more convenient that Kjeldahl
Disadvantages are
•Large dilutions are necessary for spec. reading.
•Proteins differ in the dye binding capacity
•Make standard curve based on predominant primary
amino acid present in the food.
Metode pengikatan warna (Dye
binding method)
• Gugus polar dalam protein dapat mengikat zat warna yang
bermuatan berlawanan dengan muatan pada protein
membentuk kompleks protein-zat warna yang tidak larut.
• Zat warna yang bersifat basa mengikat gugus asidik pada
permukaan protein seperti pada asam glutamat dan asam
aspartat.
• Zat warna yang memiliki gugus asam seperti COO- dan SO3akan mengikat rantai samping asam amino yang bersifat basa
seperti lisin, histidin, dan arginin.
• Zat warna yang sering digunakan adalah zat warna asidik
seperti Amido Black 10B ( max 615 nm) dan Orange G ( max
485 nm). Amido Black dan orange G bersifat asam karena
punya 2 gugus –SO3H.
HO
N
N
HO3S
SO3H
Orange G
NH2
O2N
N
OH
N
N
HO3S
N
SO3H
Amino black 10 B
DYE BINDING METHOD
DYE BINDING
Anionic dyes and Bradford Main types
Proteins will bind to certain types of dye. When this
binding
occurs,
theuse
protein-dye
will precipitate.
Anionic
dyes
principlecomplex
that proteins
bind dye
and become insoluble.
The unbound
dye isbind
thenanionic
easily determined
withataneutral
Basic groups
dye (+ charge
pH)
spectrophotometer
using a standard curve
Unbound dye inversely proportional to protein
UsingAds:
the amount
of dye
initiallyavailable
added tolysine,
the protein
quick, can
estimate
no
nasty reagents, NPN does not interfere, precise
solution,
the amount of protein can be calculated.
Disads: not sensitive, calibration curve
required for each protein, many interferences
More Protein,
more protein
Less Color
less color
Diagram sebaran yang menunjukkan plot antara kandungan nitrogen protein
terhadap metode pengikatan warna untuk 73 sampel serbuk kentang
Sumber: Wu, W.B. and Lakin, A.L. 1993. Estimation of protein in potato tissue
by dye binding. Food Chemistry 46: 49-53
BICINCHONIC ACID (BCA) METHOD
Proteins reduce cupric ions to cuprous ions under
alkaline conditions. Cuprous ions react with BCA
reagent to give a purple color.
The BCA assay primarily relies on two reactions.
• Firstly, the peptide bonds in protein reduce Cu2+ ions
from the cupric sulfate to Cu1+ . The amount of Cu2+
reduced is proportional to the amount of protein present
in the solution.
• Next, two molecules of bicinchoninic acid chelate with
each Cu1+ ion, forming a purple-colored product that
strongly absorbs light at a wavelength of 562 nm.
BICINCHONIC ACID (BCA) METHOD
Advantages
- as sensitive as Lowry but simpler
- reagent more stable than Lowry
Disadvantages
- color not stable with time  precise timing
- reducing sugars interfere more than Lowry
- color variations between proteins occur
absorbance vs concentration not absolutely
linear
Dumas Method
• Prinsip metode:
• sampel dibakar pada suhu yang sangat tinggi (700 – 800
oC). Nitrogen yang dilepaskan dianalisis secara kuantitatif
dengan gas kromatografi menggunakan detektor
konduktifitas termal. Nitrogen yang ditentukan
selanjutnya diubah menjadi kandungan protein dalam
sampel.
• Metode pembakaran Dumas ini sesuai untuk semua jenis
makanan. AOAC menggunakan metode ini untuk analisis
protein dalam daging dan dalam serealia (metode AOAC
992.15 dan metode 992.23).