1
SPECTROSCOPY
PROTEIN DETERMINATION
PHAM VAN HUNG, PhD
Introduction
Proteins are an abundant component in all
cells, and almost all except storage proteins
are important for biological functions and cell
structure.
Component?
Amino acids?
Classification?
Structure?
Functional properties?
Primary Structure of Protein
Secondary Protein Structure
Quaternary structure
A protein has size and shape as well as unique arrangement of
its polypeptide chains. (Aggregation of several peptide chains to
form a definite molecule by ionic bond, hydrogen bond, and/or
hydrophobic bond).
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SPECTROSCOPY
Importance of protein analyses
Protein analysis is important for:
Nutrition labeling
Pricing
Functional property investigation
Biological activity determination
Importance of protein analyses
Protein analysis is required when you want to
know:
1. Total protein content
2. Content of a particular protein in a mixture
3. Protein content during isolation and purification
of a protein
4. Nonprotein nitrogen
5. Amino acid composition
6. Nutritive value of a protein
Protein Determination Methods
1. Kjeldahl Method.
2 Dye Binding Method.
3. Biuret Method.
4. Lowry Method.
5. Ultraviolet Method.
Kjeldahl Method - Nitrogen Determination
(1) Digestion to conver nitrogen into an ammonium ion (NH
4
+
).
+ conc. H
2
SO
4
+ a catalyst (Copper sulfate)
(2) Neutralize NH
4
+
with NaOH to get NH
3
(3) Steam distillation of NH
3
and trap in boric acid.
(4) Titrate with hydrochloric acid.
Calculation:
Gram nitrogen/ gram of sample =
*(ml of sample - ml of blank) × N (normality) of standard acid × 0.014g/meq
weight of sample
* ml of hydrochloric acid required to titrate sample solution.
Procedure and reactions
Sample preparation
Digestion
Neutralization and Distillation
Titration
Calculations
[2]
[5]
Conversion Factors from Nitrogen to Protein for Foods
Corns Milk Whole wheat Wheat flour Nuts
Eggs Barley
Peas Oats
Meat Rye
6.25 6.38 5.83 5.70 5.30
Beans Millet
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SPECTROSCOPY
Apparatus Apparatus
Apparatus
Application
Advantages:
1. Applicable to all types of foods
2. Inexpensive (if not using an automated system)
3. Accurate; an official method for crude protein
content
4. Has been modified (micro Kjeldahl method) to
measure microgram quantities of proteins
Disadvantages:
1. Measures total organic nitrogen, not just protein
nitrogen
2. Time consuming (at least 2 h to complete)
3. Poorer precision than the biuret method
4. Corrosive reagent
Dye Binding Method
Principle:
- At low pH, basic groups of protein are (+) charged. These will
quantitatively bind a (-) charged dye.
- Proteins bind the dye to form an insoluble complex. The
unbound soluble dye is measured after equilibration of the
reaction and the removal of insoluble complex by centrifugation
or filtration.
NH
3
+
CH
CH
2
CH
2
CH
2
CH
2
N
N
H
H
C
O
C
CH
2
C
NH
+
CH
CH
N
HC
H
C
O
N
H
CH
2
CH
2
CH
2
N
H
C
NH
2
NH
2
+
Lysine
Arginine
Histidine
Acid Orange 12:
N = N
HO
SO
3
-
Procedure:
1. Mix protein, dye, buffer pH = 2.
2. Filter or centrifuge.
3. Measure absorbance of filtrate.
Dye Binding Method
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SPECTROSCOPY
Absorbance of dye bound by protein = A initial (free) die concentration - A.
filtrate die concentration
Dye Binding Method
A
b
s
o
r
b
a
n
c
e
a
t
4
7
0
n
m
% Protein (Kjeldahl)
6 8 10 12
14
16
Skim milk
1
1
2
2
x
x
x
x
Factors Influencing Dye Binding determination:
1. Temperature
2. Non-proteins.
3. Buffers systems.
4. Protein quality.
Dye Binding Method
Biuret Method
Principles: Cu
++
in alkaline solution form complexity with
peptide bonds - give pinkish-purple color.
Measure the intensity of color at 540 nm. Standard is bovine
serum albumin (BSA).
A
a
t
5
4
0
n
m
% Protein (Kjeldalh)
Lowry Method
The Lowry method combines the biuret reaction with the reduction
of the Folin–Ciocalteau phenol reagent (phosphomolybdic-
phosphotungstic acid) by tyrosine and tryptophan residues in
the proteins.
•Cu
++
in alkaline solution to form complexity with protein.
•Cu
++
catalyses oxidation of phenol group of tyrosine with
phosphomolybdic-phosphotungstic acid.
Procedure
1. Proteins to be analyzed are diluted to an appropriate
range (20–100 μg).
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 10min.
4. Freshly prepared Folin reagent is added and then the
reaction mixture is mixed and incubated at 50◦C for 10
min.
5. Absorbance is read at 650 nm.
6. A standard curve of BSA is carefully constructed for
estimating protein concentration of the unknown.
Application
Advantages:
1. Very sensitive
(a) 50–100 times more sensitive than biuret method
(b) 10–20 times more sensitive than 280-nm UV absorption method
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 h.
Disadvantages:
1. Color varies with different proteins to a greater extent than the
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 concentrations of reducing sugars, ammonium sulfate,
and sulfhydryl compounds interfere with the reaction.
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SPECTROSCOPY
Ultra-violet Absorption (UV) at 280 nm
1. Proteins show strong absorption in the region at ultraviolet
(UV) 280nm, primarily due to tryptophan and tyrosine
residues in the proteins.
Chromophoric side chains of
aromatic amino acids (Tyrosine, Tryptophan).
2. Because the content of tryptophan and tyrosine in proteins
from each food source is fairly constant, the absorbance at
280nm could be used to estimate the concentration of
proteins, using Beer’s law.
Procedure
1. Proteins are solubilized in buffer or alkali.
2. Absorbance of protein solution is read at 280nm
against a reagent blank.
3. Protein concentration is calculated according to the
equation
A =
ε
lc
where:
A = absorbance
ε
= absorptivity
l = cell or cuvette path length
c = concentration
Determination for Amino Acid Compositions of Proteins
A. Hydrolysis
1. Overnight in 6 M HCl at 100 C.
2. Enzymes.
B. Separation by ion exchange chromatography.
Mechanism of Ion-Exchange Chromatography of
Amino Acids
Na
+
Na
+
H
+
OH
-
= H
2
O
= H
2
O
OH
-
H
+
Na
+
Na
+
COO
-
H
3
N
+
Na
+
OH
COO
-
N
+
H
3
H
3
N
+
COOH
OH
COOH
COOH
H
3
N
+
So
3
-
SO
3
-
SO
3
-
So
3
-
So
3
-
SO
3
-
H
3
N
+
Exchange Resin
pH 2
pH3.5
pH4.5
LYS
HIS
ASP
GLU
ALA
VAL
LEU
pH 2.25
pH 3.25 pH4.25
Moles/Liter
Chromatogram of Amino Acids
The end!
. Factors from Nitrogen to Protein for Foods
Corns Milk Whole wheat Wheat flour Nuts
Eggs Barley
Peas Oats
Meat Rye
6.25 6. 38 5 .83 5.70 5.30
Beans Millet
3
SPECTROSCOPY. incubated at room temperature for 10 min.
3. CuSO4-K Na Tartrate-NaOH solution is added after
cooling and incubated at room temperature for 10min.
4. Freshly