PROTEIN

ANALYSIS
 LEARNING OUTCOMES
• Briefly explain definition and structure of
protein.
• State the importance of protein
determinations.
• Describe the protein analysis by using
chemical (Kjedahl method & UV- Vis
spectroscopy).
 INTRODUCTION
Proteins are polymers of amino acids
Composed of hydrogen, carbon, nitrogen, oxygen & sulfur
Twenty α-amino acids are the building blocks of proteins
Linkages between amino acids are peptide bonds – forming
polypeptide chains
Nitrogen – distinguishing element present in proteins

Proteins classified by their composition, structure,

biological function, or solubility properties
Proteins contribute to the nutritional value as well as
textural and sensory properties of foods
 INTRODUCTION
Proteins have unique conformations that could be altered
by denaturants e.g. heat, acid, alkali, 8M urea, 6M
guanidine-HCl, organic solvents and detergents
Solubility and functionality of proteins could be altered by
denaturants
Total organic nitrogen in foods represent nitrogen
primarily from proteins
Lesser extent from organic nitrogen-containing non-protein
substances

Methods to determine protein content

Basic principle – determinations of nitrogen, peptide bonds,
aromatic acids, UV absorptivity of proteins, free amino
groups, light scattering properties & dye binding capacity
IMPORTANCE OF ANALYSIS
Biological activity determination
Some proteins, including enzymes or enzyme inhibitors, are
relevant to food science & nutrition
Functional properties investigation
Proteins in various types of food have unique food functional
properties
Nutritional labeling
Protein analysis is required for :
Total protein content
Amino acid composition
Content of a particular protein in a mixture
Nutritive value (digestibilty, protein efficiency ratio, or nitrogen
balance
 CONTENT IN FOODS
Sources of proteins – animal origin and legumes
Food Item % Protein (wet wt. basis)
Dairy foods 3.5 – 35.9
Meats 12.9 – 34.3
Eggs, raw, whole 12.9
Fish 24.2 – 28.5
Cereals 0.3 – 13.3
Legumes 7.8 – 34.1
Fruits & vegetables 0.2 – 2.5
Almonds, whole 18.6
 METHODS OF PROTEIN
ANALYSIS
1. Kjedahl Method
Principle
Proteins and other organic food components in a sample
are digested with sulfuric acid in the presence of
catalysts
Total organic nitrogen is converted to ammonium sulfate;
the digest is neutralized with alkali and distilled into boric
acid solution
Borate anions are formed & titrated with standardized
acid – converted to nitrogen in the sample
Result – represents crude protein content
 METHODS OF PROTEIN
ANALYSIS
1. Kjedahl Method
Principle
Digestion by heating with sulfuric acid with the addition
of a catalyst
To complete oxidation & conversion of nitrogen to ammonium
sulfate
Digestion converts any nitrogen in the food into ammonia,
and other organic matter to CO2 & H2O
Neutralization of diluted digest
After digestion is completed, the digestion flask is connected
to a receiving flask by a tube
Solution in digestion flask is made alkaline by addition of NaOH,
which converts the ammonium sulfate into ammonia gas
 METHODS OF PROTEIN
ANALYSIS
1. Kjedahl Method
Principle
Distillation of diluted digest
(NH4)2SO4 + 2NaOH 2NH3 + 2H2O + Na2SO4
Ammonia gas is liberated from the solution, and moves out of
the digestion flask and into receiving flask (which contains an
excess of boric acid)
The low pH of the solution in receiving flask converts the
ammonia gas into the ammonium ion, and simultaneously
converts the boric acid to the borate ion

NH3 + H3BO3 (boric acid) NH4+ + H2BO3- (borate ion)

 METHODS OF PROTEIN
ANALYSIS
1. Kjedahl Method
Principle
Titration of the ammonium borate formed with standard
sulfuric / hydrochloric acid, using suitable indicator to
determine the end-point of the reaction
H2BO3- + H+ H3BO3

A reagent blank should be run to subtract reagent

nitrogen from the sample nitrogen
 METHODS OF PROTEIN
ANALYSIS
1. Kjedahl Method
Principle
A conversion factor (F) is needed to convert the measured
nitrogen concentration to a protein concentration
Most proteins contain 16% N, so conversion factor is 6.25
(equivalent to 0.16 g nitrogen per gram of protein)

% N in protein Factor
Egg or meat 16.0 6.25
Milk 15.7 6.38
Wheat 18.0 5.70
Corn 16.0 6.25
Oat 17.15 5.83
Soybean 17.51 5.71
 METHODS OF PROTEIN
ANALYSIS
1. Kjedahl Method
Calculations
1 mL 0.1M HCl = 1.4 mg N
Therefore,
1. Total nitrogen (g) per 100 g food sample
= (titre sample – titre blank) x 1.4 mg N x 100
1000 x sample weight (g)

2. Total protein (g) per 100 g food sample

= total nitrogen x factor for foodstuff analyzed
 METHODS OF PROTEIN
ANALYSIS
1. Kjedahl Method
Functions of reagents
1. Concentrated sulfuric acid
For digestion of proteins and other food components, with
the presence of catalysts to complete oxidation and
conversion of total organic nitrogen to ammonium sulfate
2. Potassium sulfate
Use to increase boiling point of sulfuric acid
Accelerate digestion mixture to shorten the reaction
3. Copper (II) sulfate
Act as catalyst
Convert organic nitrogen present to ammonium sulfate
 METHODS OF PROTEIN
ANALYSIS
1. Kjedahl Method
Functions of reagents
4. Boric acid
Use for distillation of ammonia, which contains methylene
blue & methyl red
Borate ion formed is proportional to the amount of nitrogen

Macro Kjedahl Micro Kjedahl

Sample size : 2 – 10 g Sample size : 100 – 500 mg

Flask : 500 ml Flask : 100 ml
H2SO4 : 20 – 50 ml H2SO4 : 2 – 5 ml
 METHODS OF PROTEIN
ANALYSIS
1. Kjedahl Method
Applications

Advantages
Advantages Disadvantages
Disadvantages

 Applicable
Applicable to to all
all types
types ofof food
food  Does
Does not
not give
give aa measure
measure ofof the
the true
true
 Relatively protein
protein –– measures
measures total
total organic
organic
Relatively simple
simple
nitrogen
nitrogen
 Inexpensive
Inexpensive  Different
Different proteins
proteins need
need different
different
 Accurate
Accurate andand good
good reproducibility
reproducibility correction
correction factors
factors
–– official
official method
method for for crude
crude protein
protein
content  Time
Time consuming
consuming
content
 Corrosive
Corrosive reagent
reagent
 METHODS OF PROTEIN
ANALYSIS
1. Kjedahl Method
 METHODS OF PROTEIN
ANALYSIS
Method using UV-visible spectroscopy
Methods based on UV-visible spectroscopy have been
developed to measure protein concentration
The methods use either the natural ability of proteins to
absorb (scatter) light in the UV-visible region of the
electromagnetic spectrum, or they chemically or physically
modify proteins to make them absorb (or scatter) light in
this region.
These methods requires standard curves, relating the
absorbance at a particular wavelength to the
concentration of the specific protein
Absorbance of the sample solution is measured at the
same wavelength, and its protein concentration is
determined from the calibration curve
 METHODS OF PROTEIN
ANALYSIS
2. Biuret Method
Principle
Biuret method involves a reaction with peptide linkages
Cupric ions (Cu2+) complexed with peptide bonds
(substances containing at least two (2) peptide bonds i.e.
biuret, large peptides, and all proteins) under alkaline
conditions and produced a violet-purplish colour
The absorbance of the colour produced is read at 540 nm
The colour intensity (absorbance) is proportional to the
protein content of the sample
 METHODS OF PROTEIN
ANALYSIS
2. Biuret Method
Procedure
1. Biuret reagent is mixed with a portion of protein solution of
the sample. The reagent includes copper sulfate, NaOH
and potassium sodium tartrate (to stabilize the cupric ion
in the alkaline solution)
2. The mixture is allowed to stand at room temperature for
15 – 30 min.
3. The absorbance of the mixture solution is read at 540 nm
against blank reagent.
Standard curve of [concentration] vs. absorbance is
constructed using bovine serum albumin (BSA)
 METHODS OF PROTEIN
ANALYSIS
2. Biuret Method
Applications

Determination of protein content in cereals, meat,

soybean proteins and as a qualitative test for animal feed
The method also is used widely to measure the protein
content of isolated proteins
 METHODS OF PROTEIN
ANALYSIS
2. Biuret Method

Advantages
Advantages Disadvantages
Disadvantages

 Rapid
Rapid test
test (analysis
(analysis cancan be
be  Relatively
Relatively lowlow sensitivity
sensitivity compared
compared
completed
completed within
within 30
30 min.)
min.) to
to other
other UV-vis
UV-vis methods
methods
 Colour
Colour derivations
derivations encountered
encountered  Not
Not an
an absolute
absolute method
method :: colour
colour
less
less frequently
frequently than
than other
other method
method must
must bebe standardized
standardized against
against
 Very known
known protein
protein (BSA)
(BSA) oror against
against
Very few
few substances
substances otherother than
than
proteins Kjedahl
Kjedahl nitrogen
nitrogen method
method
proteins inin foods
foods interfere
interfere with
with the
the
biuret
biuret reaction
reaction  Opalescence
Opalescence could could occur
occur in
in the
the
 Does final
final solution
solution with
with presence
presence ofof high
high
Does notnot detect
detect nitrogen
nitrogen from
from
non-peptide levels
levels of
of lipid
lipid or
or CHO
CHO
non-peptide or or non-protein
non-protein
sources
sources
 METHODS OF PROTEIN
ANALYSIS
3. Lowry Method
Principle

Lowry method combines biuret reagent with another

reagent (Folin-Ciocalteau phenol reagent) ; which reacts
with tyrosine and trytophan residues in proteins
The reaction gives a bluish colour; and the absorbance is
read at :
750 nm (high sensitivity for low protein concentration); or
500 nm (low sensitivity for high protein concentration)
 METHODS OF PROTEIN
ANALYSIS
3. Lowry Method
Procedure
1. Proteins to be analyzed is diluted to an appropriate range
(20 to 100 mg)
2. Biuret reagent is added to the diluted sample and
incubated at room temp. for 10 min.
3. Freshly prepared Folin reagent is added, mixed and
incubated
4. The absorbance of the solution is read at 650 nm
Standard curve of [concentration] vs. absorbance is
constructed using bovine serum albumin (BSA)
 METHODS OF PROTEIN
ANALYSIS
3. Lowry Method
Applications

Widely used in protein biochemistry (not widely used to

determine proteins in food systems without first
extracting the proteins from the food mixture)
 METHODS OF PROTEIN
ANALYSIS
3. Lowry Method
Applications

Advantages
Advantages Disadvantages
Disadvantages

 Very
Very sensitive
sensitive  Colour
Colour varies
varies with
with different
different proteins
proteins
 Less to
to aa greater
greater extent
extent than
than biuret
biuret
Less affected
affected by
by turbidity
turbidity of
of the
the
sample method
method
sample
 More  Colour
Colour isis not
not strictly
strictly proportional
proportional to
to
More specific
specific than
than most
most other
other
methods protein
protein concentration
concentration
methods
 Relatively  The
The reaction
reaction is
is interfered
interfered with
with
Relatively simple
simple
varying
varying degrees
degrees of of sucrose,
sucrose, lipids,
lipids,
monosaccharides,
monosaccharides, etc. etc.
 The
The reaction
reaction is
is interfered
interfered with
with high
high
concentrations
concentrations of of reducing
reducing sugars,
sugars,
ammonium
ammonium sulfate,
sulfate, andand sulfhydryl
sulfhydryl
compounds
compounds
 METHODS OF PROTEIN
ANALYSIS
4. Dye Binding Method
Principle

Protein-containing sample is mixed with a known excess

amount of anionic (negatively charged) dye in a buffered
solution (so that the proteins are positively charged)
Proteins bind the dye, to form an insoluble complex
(electrostatic attraction between the molecules)
The amount of unbound soluble dye is determined by
measuring its absorbance
The absorbance of unbound dye is measured after
equilibration of the reaction & removal of insoluble protein-dye
complex (e.g. centrifugation)
 METHODS OF PROTEIN
ANALYSIS
4. Dye Binding Method
Principle
Protein + excess dye protein-dye insoluble complex + unbound soluble dye

The anionic sulfonic acid dye, including acid orange 12,

orange G and Amido black 10B, binds cationic groups of the
basic amino acid residues and free amino terminal group of
the proteins
Unbound dye is inversely related to the protein content of the
sample
Dyebound = dyeinitial - dyefree
 METHODS OF PROTEIN
ANALYSIS
4. Dye Binding Method
Applications
The method is used to estimate proteins in milk, wheat flour,
soy products and meats

Advantages
Advantages Disadvantages
Disadvantages

 Rapid,
Rapid, inexpensive,
inexpensive, relatively
relatively  Not
Not sensitive;
sensitive; mg
mg quantities
quantities ofof
accurate
accurate proteins
proteins are
are required
required
 May
May be
be used
used toto estimate
estimate changes
changes  Proteins
Proteins differ
differ in
in basic
basic amino
amino acid
acid
in
in available
available lysine
lysine content
content ofof content,
content, soso differ
differ in
in dye-binding
dye-binding
cereal
cereal products
products during
during processing
processing capacity
capacity
 No
No corrosive
corrosive reagents
reagents  Non-protein
Non-protein components
components bind bind dye
dye
 Does
Does not
not measure
measure non-protein
non-protein (i.e.
(i.e. starch)
starch) and
and // oror protein
protein (i.e.
(i.e.
nitrogen
nitrogen calcium
calcium oror phosphate)
phosphate) –– causecause
 More error
error
More precise
precise than
than Kjedahl
Kjedahl method
method
 METHODS OF PROTEIN
ANALYSIS
SUMMARY
Kjedahl method measures organic nitrogen
Copper-peptide bond structure interactions contribute to the
analysis by the biuret and Lowry method
Amino acids are involved in the Lowry, dye-binding methods
Rapid method may be suitable for quality control purposes,
while a sensitive method is required to determine the amount
of protein in food sample