PROTEIN

ANALYSIS
Phân tích protein

GVHD: Phạm Thị Hoàn

[email protected]
 MAIN CONTENTS

Protein Content
01 Introdution in Food 03
Methods
Importance
02 of Protein
Kjeldahl, Dumas, Biuret,
Lowry , Dye-Binding 04
Methods, Bicinchoninic Acid,
Analysis Infrared Spectroscopy,
Ultraviolet 280nm Absorption
Introdution

01
INTRODUCTION
Protein Definition
Proteins are polymers of amino acids.

They are composed of elements

including hydrogen, carbon, nitrogen,
oxygen, and sulfur.
INTRODUCTION
Protein Definition
Twenty (20) different types
of amino acids occur
naturally in proteins.

Proteins differ from each

other according to the type,
number and sequence of
amino acids.
INTRODUCTION

Protein have different molecular

structures, nutritional attributes
and physiochemical properties.
INTRODUCTION

Nitrogen is the most distinguishing

element present in proteins.

Nitrogen content: 13.4 to 19.1%

INTRODUCTION
Nonprotein nitrogen could come from:
free amino acids, some vitamins,
small peptides, alkaloids,
nucleic acids, uric acid (C5H4N4O3),
phospholipids, urea, and
amino sugars, ammonium ions.
porphyrin,
INTRODUCTION

Proteins have unique conformations that could be altered by

denaturants such as:
heat,
acid, alkali,
8 M urea,
6 M guanidine-HCl,
organic solvents, and
detergents.
INTRODUCTION
Role of Protein in food
o They are a major source of
energy, as well as containing
essential amino-acids

o Proteins are also the major

structural components of
many natural foods
INTRODUCTION

Role of Protein in food

o Isolated proteins are often used in
foods as ingredients because of their
unique functional properties

o Many food proteins are enzymes

which are capable of enhancing the
rate of certain biochemical reactions
 Importance of
Protein Analysis

02
IMPORTANCE OF PROTEIN ANALYSIS
1. Nutrition labeling 2. Pricing: The cost of certain
commodities is based on the
protein content
IMPORTANCE OF PROTEIN ANALYSIS

3. Functional property investigation:

Gliadin and glutenin in wheat flour for

breadmaking,

Casein in milk for coagulation into cheese

products, and

Egg albumen for foaming

IMPORTANCE OF PROTEIN ANALYSIS

4. Biological activity determination:

The proteolytic enzymes in the

tenderization of meats

Pectinases in the ripening of fruits, and

Trypsin inhibitors in legume seeds.

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 Content
in Food

03
PROTEIN CONTENT IN FOOD
Methods

04
INTRODUCTION
The basic principles of protein assays include the
determinations of:
➢ nitrogen,
➢ peptide bonds,
➢ aromatic amino acids,
➢ dye-binding capacity,
➢ Ultraviolet/ Infrared absorptivity of proteins,
 METHODS OF PROTEIN ANALYSIS
Dumas 02 03
Biuret Method
(Nitrogen Combustion)

01
Lowry Method
Kjeldahl Method 04
ME-
Ultraviolet 280nm THODS Bicinchoninic
Absorption Method 08 05 Acid Method

Infrared Dye-Binding
Spectroscopy 07 06 Methods
Kjeldahl Method
SAMPLE Solid foods are ground to pass
PREPARATION a 20-mesh screen. Samples
for analysis should be
homogeneous.

Measure the present of

Nitrogen by method involving
PRINCIPLE digestion, neutralization,
distillation and titration.
Kjeldahl Method - Procedures
Kjeldahl Method - Procedures
Kjeldahl Method - Procedures
Kjeldahl Method - Procedures

Moles of HCl = moles of NH3 = moles of N in the sample

Kjeldahl Method - Calculation
𝑚𝑁 𝑛𝑁 × 𝑀𝑁 𝑛𝐻𝐶𝑙 × 14
%𝑁 = × 100 = × 100 = × 100
𝑚𝑠𝑎𝑚𝑝𝑙𝑒 𝑚𝑠𝑎𝑚𝑝𝑙𝑒 𝑚𝑠𝑎𝑚𝑝𝑙𝑒

𝑁𝐻𝐶𝑙 × 𝑉𝐻𝐶𝑙 × 14
= × 100
𝑚𝑠𝑎𝑚𝑝𝑙𝑒
Kjeldahl Method - Calculation

A reagent blank should be run to subtract

reagent nitrogen from the sample nitrogen.
Kjeldahl Method - Calculation
%𝑃𝑟𝑜𝑡𝑒𝑖𝑛 = %𝑁 × 6.25

Nitrogen content: 13.4 to 19.1%

 Kjeldahl Method – Alternate Procedures
In place of distillation and titration with acid, ammonia or
nitrogen can be quantitated by:
Nesslerization
4NH4OH + 2HgI2 + 4KI + 3KOH → NH2Hg2IO + 7KI + 2H2O
ammonium dimercuric iodide,
red-orange, 440 nm
Kjeldahl Method – Alternate Procedures
In place of distillation and titration with acid, ammonia or
nitrogen can be quantitated by:
NH3 + phenol + hypochloride → OH− indophenol
(blue, 630nm)
Kjeldahl Method – Alternate Procedures
➢ Direct measurement of ammonia,
using ion chromatographic method

In place of titration, ammonia or

nitrogen can be quantitated by:

➢ pH measurement after distillation

into known volume of boric acid
 Kjeldahl Method

Measures total 01 Applicable to all

organic nitrogen 01 types of food
Disadvan
Time cosuming 02 -tages 02 Inexpensive
(at least 2hr)
Advan- 03 Accurate
Poorer precision than 03
the bjuret method tages
Has been modified
04
Corrosive reagent
04 Micro Kjeldahl Method
 METHOD OF PROTEIN ANALYSIS
Dumas 02 03
Biuret Method
(Nitrogen Combustion)

01
Lowry Method
Kjeldahl Method 04
ME-
Ultraviolet 280nm THODS Bicinchoninic
Absorption Method 08 05 Acid Method

Infrared Dye-Binding
Spectroscopy 07 06 Methods
Dumas (Nitrogen Combustion) Method

PRINCIPLE
Dumas Method - Applications

Suitable for all types of foods.

AOAC Method 992.15 is for meat and

AOAC Method 992.23 is for cereal grains
Dumas Method
01 Requires no
hazardous chemicals
01 Expensive
equipment
02 Rapid ( 3 min)

Measures
02 total organic
Can analyze up
03 to 150 samples
nitrogen
without attention

Advantages Disadvantages
 METHOD OF PROTEIN ANALYSIS
Dumas 02 03
Biuret Method
(Nitrogen Combustion)

01
Lowry Method
Kjeldahl Method 04
ME-
Ultraviolet 280nm THODS Bicinchoninic
Absorption Method 08 05 Acid Method

Infrared Dye-Binding
Spectroscopy 07 06 Methods
Biuret Method

PRINCIPLE
A violet-purplish color is
produced when cupric ions
are complexed with peptide
bonds under alkaline
conditions

The absorbance of the color

produced is read at 540 nm.
Biuret Method - Procedure
1 mL mẫu
A 5-ml Biuret reagent is mixed 5 mL BR
01 with a 1-ml portion of protein
solution (1–10mg protein/ml)
After the reaction mix is
02 allowed to stand at room
temperature for 15 or 30min

The absorbance is read at

03 540nm against a reagent blank
 Biuret Method - Procedure

The Biuret reagent includes

water, copper sulfate, NaOH,
and potassium sodium
tartrate (NaKC4H4O6.4 H2O),
which is used to stabilize the
cupric ion in the alkaline
solution.
Biuret Method - Procedure
3. Filtration or centrifugation
before reading absorbance if the
reaction mixture is not clear.
Biuret Method - Procedure
A standard curve of
concentration versus
absorbance is
constructed using bovine
serum albumin (BSA).
Biuret Method - Application
Used to determine proteins in cereal, meat, soybean
proteins, and as a qualitative test for animal feed [AOAC
Method 935.11]
Biuret Method
Advantages

Less expensive; Color

rapid (30 min); 01 02 deviations
simplest

Very few
Detect nitrogen
04 substances
from protein 03 interfere with the
sources
biuret reaction
Biuret Method
Disadvantages
Not an absolute Not very sensitive
method (2–4 mg protein)
06 01

Opalescence Bile pigments

could occur 05 02 → absorbance

Color varies Ammonium salts

with different 04 03 interfere with the
proteins reaction
 METHOD OF PROTEIN ANALYSIS
Dumas 02 03
Biuret Method
(Nitrogen Combustion)

01
Lowry Method
Kjeldahl Method 04
ME-
Ultraviolet 280nm THODS Bicinchoninic
Absorption Method 08 05 Acid Method

Infrared Dye-Binding
Spectroscopy 07 06 Methods
Lowry Method
The method combines the biuret
reaction with the reduction of the
Folin–Ciocalteau phenol reagent by
tyrosine and tryptophan residues in
the proteins

The bluish color developed is read at

750nm (for low protein concentration)
or 500nm (for high protein
concentration).
 Lowry Method - Procedure
Adapted From Hartree, 1973

01 02 03 04 05 06

Folin Measure Estimate

Dilute KNaTartr Biuret
reagent is absorbance protein
proteins ate- solution is
added → at 650 nm concentration
(20–100 Na2CO3 added → mixed → of sample
µg)
solution is incubated incubated using a
added → at 50°C standard
curve of BSA
incubated for 10 min.
 Lowry Method - Application
The Lowry method
has been widely used
in protein biochemistry

In food systems,
proteins need to be
extracted from the
food mixture.
 Lowry Method

Less affected
Very sensitive 01 02 by turbidity of
the sample

Advan-
tages
More specific
Relatively simple
(1–1.5 hr) 04 03 than most other
methods.
 Lowry Method
Color is not strictly
proportional to
02 protein
concentration
Color varies
with different 01 Disadvan-
proteins
tages

The reaction is
03 interfered
 METHOD OF PROTEIN ANALYSIS
Dumas 02 03
Biuret Method
(Nitrogen Combustion)

01
Lowry Method
Kjeldahl Method 04
ME-
Ultraviolet 280nm THODS Bicinchoninic
Absorption Method 08 05 Acid Method

Infrared Dye-Binding
Spectroscopy 07 06 Methods
 Bicinchoninic Acid Method
PRINCIPLE

●Protein reduce cupric ions to cuprous ions under

alkaline conditions; cuprous ions react with BCA
reagent to give purple color (absorb at 562 nm)
 Bicinchoninic Acid Method Estimate
protein
Mix (one step) the
concentration
protein solution with
Read the in the sample
the BCA reagent
absorbance at
Construct
562 nm against
01 37°C for 30 min, a standard 05
a reagent blank
Room temperature for 2 h, curve
60°C for 30 min. using BSA
03

02 04

Incubate

PROCEDURE
Bicinchoninic Acid Method

BCA reagent contains BCA sodium salt, sodium carbonate,

NaOH, and copper sulfate, pH 11.25.

BCA method has been used in protein isolation and purification

 Bicinchoninic Acid Method
ADVANTAGES

Sensitivity 02 One-step mixing

01 03 The reagent is
Medium concentrations
stable
of denaturing reagents
(4 M guanidine-HCl or
3 M urea) do not Nonionic detergent and
interfere
05 04 buffer salts do not
interfere with the reaction
Bicinchoninic Acid Method
DISADVANTAGES
Any compound
Color is not capable of reducing
stable with time 01 02 Cu+2 to Cu+ will lead
to color formation

Reducing
Color variations sugars interfere
among proteins 04 03 to an extent
 METHOD OF PROTEIN ANALYSIS
Dumas 02 03
Biuret Method
(Nitrogen Combustion)

01
Lowry Method
Kjeldahl Method 04
ME-
Ultraviolet 280nm THODS Bicinchoninic
Absorption Method 08 05 Acid Method

Infrared Dye-Binding
Spectroscopy 07 06 Methods
Dye-Binding Methods

Anionic Dye-Binding Method

Bradford Dye-Binding Method
Anionic Dye-Binding Method
PRINCIPLE
Protein + excess dye → Protein-dye insoluble complex +
unbound soluble dye
Anionic Dye-Binding Method
PROCEDURE

Add the sample to an Shake the

01 excess dye solution with 02 content → filter
known concentration. or centrifuge

Measure the absorbance of the unbound

03 dye solution → Estimate dye
concentration from a dye standard curve.
Anionic Dye-Binding Method
PROCEDURE

Construct a straight calibration curve

between the unbound dye concentration
04 and total nitrogen

Estimate the protein content of the

05 unknown sample
Anionic Dye-Binding Method
APPLICATION

Used to estimate proteins in milk, wheat flour, soy

products, and meats.
Anionic Dye-Binding Method

Advantages
1. Rapid (15 min or less), inexpensive, and relatively accurate
2. No corrosive reagents.
3. Measure protein nitrogen.
4. Precise
5. May be used to estimate the changes in available lysine
content of cereal products during processing
Anionic Dye-Binding Method
Disadvantages
1. Not sensitive; (mg pr.)
2. Proteins differ in basic amino acid content and so differ in dye-
binding capacity.
3. Not suitable for hydrolyzed proteins
4. Some nonprotein components bind dye (starch) or protein
(calcium or phosphate)
Bradford Dye-Binding Method
PRINCIPLE

●Protein are bound by a Coomassie brilliant blue G-250 dye

changing color from reddish to blue (bluish) shifting maximum
absorbance from 465 to 595 nm

●Based on the amphoteric nature of proteins

Bradford Dye-Binding Method
PROCEDURE
Mix the samples Construct a
solutions with the standard curve
Bradford reagent using BSA

Prepare Bradford reagent Read the Estimate protein

(Coomassie Brilliant Blue absorbance at concentration in
G-250 is dissolved in 95% 595 nm against a the sample
ethanol and acidified with reagent blank
85% phosphoric acid.)
Bradford Dye-Binding Method

APPLICATION

The Bradford method has been used successfully to determine

protein content in worts, beer products and in potato tubers.

Measures protein or peptides with molecular mass approximately

equal to or greater than 4000 Da
 Bradford Dye-Binding Method

Rapid (2 min)
01 02 Reproducible

Advan-
tages
No interference 04 03 Sensitive
 Bradford Dye-Binding Method

Color varies with

02 different types of
proteins

Disadvan-
tages
The protein–dye
complex can bind 01
to quartz cuvettes
 METHOD OF PROTEIN ANALYSIS
Dumas 02 03
Biuret Method
(Nitrogen Combustion)

01
Lowry Method
Kjeldahl Method 04
ME-
Ultraviolet 280nm THODS Bicinchoninic
Absorption Method 08 05 Acid Method

Infrared Dye-Binding
Spectroscopy 07 06 Methods
Infrared Spectroscopy
Infrared spectroscopy measures
the absorption of radiation (near-
or mid-infrared regions) by
molecules in food or other
substances.

Different functional groups in a

food absorb different frequencies
of radiation.
Infrared Spectroscopy
For proteins and peptides,
various bands (e.g., 3300–
3500 nm; 2080–2220 nm;
1560–1670 nm) characteristic
of the peptide bond can be
used to estimate the protein
content of a food.
Infrared Spectroscopy
Application
Infrared Milk Analyzers; Near-
infrared spectroscopy is applicable
to a wide range of food products
(e.g., grains, cereal, meat, and
dairy products)

Infrared Milk Analyzers

 Infrared Spectroscopy

Instruments are
Rapid (30s to 2 min)
expensive

DIS ADVAN-
TAGES

They must be
Minimal training
calibrated properly
 METHOD OF PROTEIN ANALYSIS
Dumas 02 03
Biuret Method
(Nitrogen Combustion)

01
Lowry Method
Kjeldahl Method 04
ME-
Ultraviolet 280nm THODS Bicinchoninic
Absorption Method 08 05 Acid Method

Infrared Dye-Binding
Spectroscopy 07 06 Methods
Ultraviolet 280nm
Absorption Method
Proteins show strong absorption in
the region at ultraviolet (UV) 280
nm, primarily due to tryptophan and
tyrosine residues in the proteins.

The content of tryptophan and

tyrosine in proteins from each food
source is fairly constant
Ultraviolet 280nm
Absorption Method
Application

The UV 280-nm method has been used to determine the protein

contents for milk and meat products
 Ultraviolet 280nm
Absorption Method
No interference
from ammonium
02 sulfate and other
buffer salts
Rapid and
relatively 01 Advan-
sensitive
tages

03 Nondestructive
 Ultraviolet 280nm
Absorption Method Aromatic amino
acid contents in
Nucleic acids
the proteins from
also absorb at 01 02 various food
280 nm
sources differ
Dis- considerably
advan-
A relatively tages The solution
pure system is
required to use 04 03 must be clear
and colorless
this method
 SUMMARY

Methods based on the unique characteristics of proteins and

amino acids.

01 The Kjeldahl and Dumas methods measure nitrogen.

02 Infrared spectroscopy is based on absorption of a

wavelength of infrared radiation specific for the peptide bond.
 SUMMARY

Methods based on the unique characteristics of proteins and

amino acids.
03 Copper–peptide bond interactions contribute to the
analysis by the Biuret and Lowry methods.
04 Amino acids are involved in the Lowry, dye-binding,
and UV 280nm methods.
 SUMMARY

Methods based on the unique characteristics of proteins and

amino acids.
05 The BCA method utilizes the reducing power of
proteins in an alkaline solution.

→ The various methods differ in their speed and sensitivity.

 SUMMARY
Rapid methods may be suitable for quality control purposes,
while a sensitive method is required for work with a minute
amount of protein.

Indirect colorimetric methods (Anionic dye-binding) usually

require the use of a carefully selected protein standard or a
calibration with an official method (e.g., Kjeldahl).
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