a review on general methods of analysis of proteins

www.wjpps.com Vol 6, Issue 11, 2017.

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Siddiqui. World Journal of Pharmacy and Pharmaceutical Sciences

A REVIEW ON GENERAL METHODS OF ANALYSIS OF PROTEINS

Juveriya Fatima Siddiqui*

Deccan School of Pharmacy Aghapura, Nampally Hyderabad.

ABSTRACT

proteins are polymers of amino acids. Twenty different types of amino

acids occur naturally in proteins. Proteins differ from each other

according to the type, number and sequence of amino acids that make

up the polypeptide backbone. As a result they have different molecular

structures, nutritional attributes and physiochemical properties.

Proteins are important constituents of foods for a number of different

reasons. They are a major source of energy, as well as containing

essential amino-acids, such as lysine, tryptophan, methionine, leucine,

isoleucine and valine, which are essential to human health, but which

the body cannot synthesize. Proteins are also the major structural components of many

natural foods, often determining their overall texture, e.g., tenderness of meat or fish

products. Isolated proteins are often used in foods as ingredients because of their unique

functional properties, i.e., their ability to provide desirable appearance, texture or stability.

Typically, proteins are used as gelling agents, emulsifiers, foaming agents and thickeners.

Many food proteins are enzymes which are capable of enhancing the rate of certain

biochemical reactions. These reactions can have either a favorable or detrimental effect on

the overall properties of foods. Food analysts are interested in knowing the total

concentration, type, molecular structure and functional properties of the proteins in foods.

KEYWORDS: Proteins, Qualitative Methods, Quantitative Methods.

INTRODUCTION

The word PROTEIN comes from Greek language (prota) which means "of primary

importance. Proteins are large biological molecules with molecular weight up to few million

Daltons. Proteins are made of amino acids linked into linear chains, called polypeptide

chains. Amino acids links between each other by peptide bonds - this peptide bond is formed

between the carboxyl and amino groups of neighbouring amino acids. In this way they can

WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

SJIF Impact Factor 6.647

Volume 6, Issue 11, 597-619 Review Article ISSN 2278 – 4357

Article Received on

15 September 2017,

Revised on 06 Oct. 2017, Accepted on 27 Oct. 2017,

DOI: 10.20959/wjpps201711-10468

*Corresponding Author

Juveriya Fatima Siddiqui

Deccan School of Pharmacy

Aghapura, Nampally

Hyderabad.

http://www.wjpps.com/

http://proteincrystallography.org/amino-acid/


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make large, chain like molecules called polymers, which may contain as few as two or as

many as 3,000 amino-acid units. If there are more than 10 units in a chain, the chain is called

a polypeptide, while a chain with 50 or more amino-acid units is known as a protein.

The sequence of the polypeptide chain is defined by a gene with genetic code. There are

only 20 standard amino acids that exist in living organism. In total the number of different

proteins, which it is possible to produce from 20 amino acids is enormous. It is almost

impossible to estimate the total number of different proteins in the nature. For example only

in E. coli cell about 3000 different proteins are known.

Most of us recognize the term protein in a nutritional context as referring to a class of foods

that includes meats, dairy products, eggs, and other items. Certainly, proteins are an

important part of nutrition, and obtaining complete proteins in one's diet is essential to the

proper functioning of the body. But the significance of proteins extends far beyond the dining

table. “Proteins are integral to the formation of DNA, a molecule that contains genetic codes

for inheritance, and of hormones.

Proteins are of importance in all biological systems, playing a wide variety of structural and

functional roles. They form the primary organic basis of structures such as hair, tendons,

muscle, skin, and cartilage. All of the enzymes, the catalysts in biochemical transformations,

are protein in nature. Many hormones, such as insulin and growth hormone, are proteins. The

substances responsible for oxygen and electron transport (hemoglobin and the cytochromes,

respectively) are conjugated proteins that contain a metalloporphyrin as the prosthetic group.

Chromosomes are highly complex nucleoproteins, that is, proteins conjugated with nucleic

acid. Viruses are also nucleoprotein in nature. Of the more than 200 amino acids are present

in mammalian proteins. Thus, proteins play a fundamental role in the processes of life.[1]

Definition

Proteins are a large complex organic compounds and are composed mostly of amino acids

linked with peptide bonds

Proteins differ from each other according to the type, number and the sequence of amino

acids that make up the polypeptide backbone.

Proteins are important constituents of foods for a number of different reasons.

They are a major source of energy as well as containing essential amino acids.

Proteins are polymers of amino acids


http://proteincrystallography.org/amino-acid/

http://www.answers.com/topic/hormone

http://www.answers.com/topic/cartilage

http://www.answers.com/topic/insulin

http://www.answers.com/topic/hormone

http://www.answers.com/topic/hemoglobin

http://www.answers.com/topic/metalloporphyrin

http://www.answers.com/topic/prosthetic

http://www.answers.com/topic/nucleic-acid

http://www.answers.com/topic/nucleic-acid

http://www.answers.com/topic/nucleoprotein


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proteins differ from each other according to the type, number and sequence of amino acids

that make up the polypeptide backbone. As a result they have different molecular structures,

nutritional attributes and physiochemical properties.

STRUCTURES OF PROTEINS

1. Primary structure

2. Secondary structure

3. Tertiary structure

4. Quaternary structure

PRIMARY STRUCTURE

It is a linear sequence of amino acids joined together by peptide bonds.

It is a simple and unfolded structure of polypeptide chains.

SECONDARY STRUCTURE

The primary structure of protein folds to forms secondary structure.

It is a regular, rigid and tubular.

TERTIARY STRUCTURE

Two or more secondary structure combines to form a tertiary structure .

It is a three dimensional folding structure by complete folding of the sheets and helices of

a secondary structure.

QUATERNARY STRUCTURE

It refers to the way individual polypeptides combines to form complexes quaternary

structure.[2]

Classification of Proteins: All proteins contain nitrogen, carbon, hydrogen, oxygen, sulphur;

some contain also phosphorus and iron. However, there are many forms of protein, all

possessing widely varied chemical and physical properties.[3]


http://chestofbooks.com/food/science/Domestic-Science-School/Chapter-VIII-Protein-Eggs.html

http://chestofbooks.com/food/science/Domestic-Science-School/Iron.html


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SUMMARY OF CLASSIFICATION OF PROTEINS

GENERAL METHOD OF ANALYSIS OF PROTEINS

1. Qualitative analysis of proteins

All proteins do not contain the same amino acids. The various amino acid constituents of

proteins may be identified by various color reactions. Based up on physical and chemical

properties and the presence of different amino acids, proteins in a given solution can be

analysed under the following headings.

I. Color reaction of proteins.

II. Precipitation reaction of proteins.

I.Color reactions of proteins

General tests for proteins

a) Biuret test

Principle

It detects the presence of peptide bonds.

Cupric ions in alkaline medium react with nitrogen’s of the peptides and proteins to form

a violet/purple color complex.

The reaction is so named since biuret (NH2–CONH–CONH2 ) is formed when two

molecules of urea condensed at 1800C.

The minimum requirement for a positive test is the presence of 2 peptide bonds in the

molecule.



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Reaction

Procedure

To the 2ml of sample solution and 3ml of 10% NaOH add 4-5 drops of 0.5% copper

sulphate solution, mix well. Appearance of violet color. Peptides and proteins are present.

Application: It is a common and delicate test for the identification of protein in a biological

material.

b) Ninhydrin test

Principle

Ninhydrin (triketohydrindene hydrate), powerful oxidizing agent, reacts with the α-amino

group of amino acid or proteins and undergoes oxidative decarboxylation in the presence of

heat to form a purple-violet color complex.

This is one of the sensitive test and is used for estimation of amino acids.

Ninhydrin is reduced to hydridantin during reaction with α-amino acid. The amino acid is

converted to aldehyde, ammonia and carbon dioxide.

Hydridantin reacts with liberated ammonia and another molecule of ninhydrin to form a

purple/violet color complex.

Reaction



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Procedure

To 1ml of test solution add 2 drops of ninhydrin reagent and boil. Appearance of purple

color.

c) Xanthoproteic test

Principle

Certain substituted benzene compounds react with nitrous acid in presence of heat and

undergoes nitration, to form yellow colored nitro/nitroso compounds. These nitro compounds

in alkaline medium ionize freely and produce deep yellow/orange color.

Tyrosine, Tryptophan, Phenylalanine contain substituted benzene ring and are responsible

for the positive test given by most proteins.

Reaction

Procedure

To 2ml of test solution add 1ml of concentrated HNO3. Heat the solution for 2min. cool

under tap water. Note the yellow color in acid medium. Divide the contents into two parts. To

half of the yellow solution add 40% NaOH till the solution is alkaline to litmus. Observe the

orange color.[4]

d) Millon’s test(Hoffman’s Reaction)

Principle

The test is answered by hydroxy phenyl group. Tyrosine and protein containing tyrosine

will give red colored complex with million’s reagent. The color is probably due to the

formation of a mercuric phenolate ion with the nitrated phenol radical in tyrosine.

Reaction



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Procedure

To 1ml of test solution add 1ml of mercuric sulphate. Boil gently for 30 seconds. Add 2

drops of 1% sodium nitrite

E) Hopkins Cole test

Principle

The hopkins coles reaction, also known as the glyoxylic acid reaction,is a chemical test

used for detecting the presence of tryptophan in protein.

concentrated H2SO4 causes dehydration of tryptophan which reacts with Hopkins Cole

reagent to give a reddish violet ring between two layers.

Procedure

Add 1 ml of original solution (protein solution) in a test tube.

Add 1 ml of Hopkins-Cole reagent in the test tube which consists of glyoxylic acid.

Mix thoroughly.

Then add 1 ml of concentrated sulphuric acid, pouring it down along the side of the test

tube.

Indication

A deep violet or purple ring forms at the junction of the two liquids. This indicates the

presence of tryptophan.[5]

Reaction

I. Precipitation reactions of proteins

Principle

Proteins are precipitated either by neutral salts like ammonium sulphate or heavy metals (e.g.

HgCl2, AgNO3,CuSO4) 0r alkaloidal reagents [e.g. picric acid, phosphotungstic acid, and

tannic acid, or dehydrating agents (e.g. alcohol and acetone)].



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Proteins exist in collaidal solution due to hydration of polar groups(-COO,-NH3+,OH)

They can be precipitated by dehydration or neutralization of polar groups.

1) Precipitation by neutral salt(ammonium sulphate)

Principle: Neutral salts like ammonium sulphate precipitate proteins by neutralization of

charges on the proteins and dehydration.

Half saturation

To 3 ml of sample, add equal volumes of saturated NH4SO4 solution. Mix and allow to

stand. Precipitate indicates presence of globulin. Filter it, dissolve the residue (ppt) in saline

and perform. Biuret test using 40% NAOH. If it is positive that indicates globulin precipitated

by half saturation.

Full saturation test

To the 3ml of sample, add solid ammonium sulphate until the solution is saturated. Mix and

allow to stand. A white precipitate indicates the presence of albumin. Filter it. Dissolve the

precipitate in water and perform the Biuret test using 40% NAOH. If it is positive, it confirms

that the precipitate is due to albumin.

2) Addition of organic solvents like alcohol

Principle

Alcohol is a dehydrating agent when alcohol is added to protein solution, it reduces the

amount of water needed to keep protein in solution. This results in precipitation of protein.

Procedure

1ml sample+1ml alcohol. Mix and let stand. A white ppt occurs.

3) Precipitation by alkaloidal reagents

Principle

In acidic medium a protein has positive charges. –ve charge of picrate neutralizes the

positive charge on protein and form the ppt of protein picrate.

Procedure

1ml albumin + 1ml picric acid yellow ppt is formed



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4) Precipitation by heavy metalsalts

Principle

In alkaline medium proteins have negative charges. The positive charge of lead neutralizes

the negative charge of protein and forms the ppt of lead proteinate.

Procedure

2ml sample + 2-3 drops of basic lead acetate white ppt is formed

5) Precipitation by heat coagulation test

Principle

Proteins are easily denatured when subjected to heat treatment. In case of albumin the

denaturation is followed by coagulation.

Procedure

Take 3/4th

of sample in a test tube. Hold the tube over a flame in a slanting position and boil

the upper half of solution. The lower half serves as control. A cloudy white color will be

observed in the heated portion. Add a few drops of 1% acetic acid.Coagulation takes place

and albumin is ppt.

6) Precipitation by acids

Principle

Conc. Acid causes denaturation of proteins, which brings native proteins into insoluble acid

Meta proteins. Derived proteins like gelatin and peptone are not sufficiently denatured by

acids and thus don’t get ppt.

Procedure

To 1ml of protein solution in test tube, add few drops of 1% acetic acid,white precipitate is

formed.[6]

2. QUANTITATIVE METHODS FOR ANALYSIS OF PROTEINS

Kjeldahl method

Enhanced Dumas method

Methods using UV-visible spectroscopy

Biuret Method

Lowry Method



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Dye binding method

Turbidimetric method

Bradfordmethod

Bicinchonic acid method.

1. Kjeldahl method

Nitrogen is one of the five major elements found in organic materials such as protein. This

fact was recognized by a Danish chemist, Johan Kjeldahl, who used it as a method of

determining the amount of protein in samples taken from a wide variety of organisms. In

1883 Kjeldahl presented to the Danish Chemical Society a method (much revised since his

day) for determining the amount of nitrogen in mixtures of substances containing ammonium

salts, nitrate, or organic nitrogen compound.

Principle

In the Kjeldahl procedure, proteins and other organic food components in a sample are

digested with sulfuric acid in the presence of catalysts. The total organic nitrogen is

converted to ammonium sulfate.The digest is neutralized with alkali and distilled into a boric

acid solution. The borate anions formed are titrated with standardized acid, which is

converted to nitrogen in the sample. The result of the analysis represents the crude protein

content of the food since nitrogen also comes from nonprotein components.

In 1883, Johann Kjeldahl developed the basic process of today’s Kjeldahl method to analyze

organic nitrogen.

General steps in the original method include the following: 1. Digestion with sulfuric acid,

with the addition of powdered potassium permanganate to complete oxidation and conversion

of nitrogen to ammonium sulfate. 2. Neutralization of the diluted digest, followed by

distillation into a known volume of standard acid, which contains potassium iodide and

iodate. 3. Titration of the liberated iodine with standard sodium thiosulfate.

Generalprocedure

Sample preparation: Solid foods are ground to pass a 20-mesh screen. Samples for analysis

should be homogeneous. No other special preparations are required.

Kjeldahl method



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A) Digestion

Place sample (accurately weighed) in a Kjeldahl flask. Add acid and catalyst; digest until

clear to get complete breakdown of all organic matter.Nonvolatile ammoniumsulfate is

formed from the reaction of nitrogen and sulfuric acid.

Protein −−−−−−−→(NH4)2 SO2 [1]

During digestion, protein nitrogen is liberated to form ammonium ions; sulfuric acid oxidizes

organic matter and combines with ammonium formed; carbon and hydrogen elements are

converted to carbon dioxide and water.

B) Neutralization and digestion

The digest is diluted with water. Alkali-containing sodium thiosulfate is added to neutralize

the sulfuric acid. The ammonia formed is distilled into a boric acid solution containing the

indicators methylene blue and methyl red.

(NH4) 2SO4+2NaOH→2NH3 +Na2 SO4 +2H2 O [2]

NH3 +H3 BO3 (boric acid)→NH4+H2 BO3- (borate ion) [3]

C) TiTRATION

Borate anion (proportional to the amount of nitrogen)is titrated with standardized HCl.

H2 BO3 - +H+ →H3 BO3 [4]

The concentration of hydrogen ions (in moles) required to reach the end-point is equivalent to

the concentration of nitrogen that was in the original food (Equation 3). The following

equation can be used to determine the nitrogen concentration of a sample that weighs m

grams using a xM HCl acid solution for the titration.

[5]

Where vs and vb are the titration volumes of the sample and blank, and 14g is the molecular

weight of nitrogen N. A blank sample is usually ran at the same time as the material being

analyzed to take into account any residual nitrogen which may be in the reagents used to

carry out the analysis. Once the nitrogen content has been determined it is converted to a

protein content using the appropriate conversion factor: %Protein = F� %N.



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Advantages

The Kjeldahl method is widely used internationally and is still the standard method for

comparison against all other methods

1. Its universality, high precision and good reproducibility have made it the major method for

the estimation of protein in foods.

2. Applicable to all types of foods.

3. Inexpensive(if not using an automated system)

4. Accurate;an official method for crude protein content

5. Has been modified(micro kjeldahl method) to measure microgram quantities of proteins.

Disadvantages

1. It does not give a measure of the true protein, since all nitrogen in foods is not in the form

of protein. Different proteins need different correction factors because they have different

amino acid sequences.

2. The use of concentrated sulphuric acid at high temperatures poses a considerable hazard, as

does the use of some of the possible catalysts the technique is time consuming to carry-out.

measure total organic nitrogen,not just protein nitrogen.

3. Time consuming*(at least 2hrs to complete)

4. Poorer precision.than the biuret method.

2. Enhanced Dumas method:

Recently, an automated instrumental technique has been developed which is capable of

rapidly measuring the protein concentration of food samples. This technique is based on a

method first described by a scientist called Dumas over a century and a half ago. It is

beginning to compete with the Kjeldahl method as the standard method of analysis for

proteins for some foodstuffs due to its rapidness.

General Principles

A sample of known mass is combusted in a high temperature (about 900°C) chamber in the

presence of oxygen. This leads to the release of C02, H20 and N2. The C02& H20 are

removed by passing the -gasses over special columns that absorb them. The nitrogen content

is then measured by passing the remaining gasses through a column that has a thermal

conductivity detector at the end. The column helps separate the nitrogen from any residual

C02 and H20 that may have remained in the gas stream. The instrument is calibrated by

analyzing a material that is pure and has a known nitrogen concentration, such as EDTA (=



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9.59%N). Thus the signal from the thermal conductivity detector can be converted into

nitrogen content. As with the Kjeldahl method it is necessary to convert the concentration of

nitrogen in a sample to the protein content, using suitable conversion factors which depend

on the precise amino acid sequence of the protein.

Advantages and Disadvantages

Advantages: It is much faster than the Kjeldahl method (under 4 minutes per measurement,

compared to 1-2 hours for Kjeldahl). It doesn't need toxic chemicals or catalysts. Many

samples can be measured automatically. It is easy to use.

Disadvantages: High initial cost. It does not give a measure of the true protein, since all

nitrogen in foods is not in the form of protein. Different proteins need different correction

factors because they have different amino acid sequences. The small sample size makes it

difficult to obtain a representative sample.[6]

.

3. UV-visible spectroscopy method: A number of methods have been devised to measure

protein concentration, which are based on UV-visible spectroscopy. These methods use either

the natural ability of proteins to absorb (or 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. The basic principle behind each of these tests is

similar. First of all a calibration curve of absorbance (or turbidity) versus protein

concentration is prepared using a series of protein solutions of known concentration. The

absorbance (or turbidity) of the solution being analyzed is then measured at the same

wavelength, and its protein concentration determined from the calibration curve. The main

difference between the tests are the chemical groups which are responsible for the absorption

or scattering of radiation, e.g., peptide bonds, aromatic side-groups, basic groups and

aggregated proteins.

These methods use either the natural ability of proteins to absorb or scatter the light in the uv

visible region of the electromagnetic spectrum or they chemically or ohysically modify

proteins to make them absorb light in this region A number of the most commonly used UV-

visible methods for determining the protein content of foods are highlighted below:

A. DIRECT MEASUREMENT AT 280NM

B. BIURET METHOD

C. LOWRY METHOD



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D. DYE BINDING METHODS

E. TURBIDIMETRIC METHOD.

a) Direct measurement at 280nm

Tryptophan and tyrosine absorb ultraviolet light strongly at 280 nm. The tryptophan and

tyrosine content of many proteins remains fairly constant, and so the absorbance of protein

solutions at 280nm can be used to determine their concentration. The advantages of this

method are that the procedure is simple to carry out, it is non destructive, and no special

reagents are required. The major disadvantage is that nucleic acids also absorb strongly at

280 nm and could therefore interfere with the measurement of the protein if they are present

in sufficient concentrations. Even so, methods have been developed to overcome this

problem, e.g., by measuring the absorbance at two different wavelengths.

b) Biuret Method

• A violet-purplish color is produced when cupric ions (Cu2+) interact with peptide bonds

under alkaline conditions.

• The biuret reagent, which contains all the chemicals required to carry out the analysis, can

be purchased commercially.

• A 5ml biuret reagent is mixedwith a 1 ml portion of protein solution

• The reagent includes copper sulfate,NAOH potassium sodium tartarate,which is used to

stabilize the cupric ion in the alkaline solution.

• After the reaction mixture is allowed to stan at room temperature for 15-30mins,the

absorbance is read at 540 nm.

• Filteration or centrifugation before reading absorbance is required if the reaction mixture is

not clear a standard curve of concentration vs absorbance is constructed using bovine serum

albumin.

Applications

• This method has been used to determine proteins in cereal,meat,soybean proteins.

• This method can also be used to measure the protein content of isolated proteins

Advantages

1. Less expensive than the kjeldahl method;rapid;simplest method of analysis of proteins.

2. Colour deviations are encountered less frequently than with lowry,uv absorption or

turbidimetric methods.



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3. Very few substances other than proteins in food interfere with the biuret reaction.

4. Doesnot detect nitrogen from nonpeptide or nonprotein sources.

Disadvantages

1. Not very sensitive as comparedto the lowry method; requires atleast 2-4 mg protein for

assay.

2. Absorbance could be contributed from the bile pigments if present.

3. High concentration of ammonium salts interfere with the reaction.

4. Colour varies with different proteins; gelatin givesa pinkish purple colour.

5. Opalescence could occur in the final solution if high levels of lipids or carbohydrates are

present.

6. Not an obsolute method;colour must be standardized against known protein or against the

kjeldahl nitrogen method.

c) Lowry Method: The Lowry method combines the biuret reagent with another reagent (the

Folin-Ciocalteau phenol reagent) which reacts with tyrosine and tryptophan residues in

proteins. This gives a bluish color which can be read somewhere between 500 - 750 nm

depending on the sensitivity required. There is a small peak around 500 nm that can be used

to determine high protein concentrations and a large peak around 750 nm that can be used to

determine low protein concentrations. This method is more sensitive to low concentrations of

proteins than the biuret method.

Procedure

• Proteins to be analysed are diluted to an appropriate range in the concentration of 20-

100mg.

• Sodium or potassium tartarate is mixed with sodium carbonate solution which is added at

room temperature and mixed for 10 minutes.

• Later copper sulphate is added and finally folent reagent is added to the protein solution.

• The contents are mixed well and the absorbance of the resulting solution is read at 650nm.

• A standard curve for bovine serum albumin is carefully constructed for estimating protein

concentration.

Applications

Because of its simplicitiy and sensitivity;the lowrty method has been used in protein

biochemistry.



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Advantages

1. Very sensitive

2. Less effected by the turbidity of the sample.

3. More specific than most other methods.

4. Relatively simple;can be done in 1-1.5 hrs.

Disadvantages

1. Colour varieswith different proteins to a greater extent than the biuret method.

2. Colour 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.

d) Dye binding method

A known excess of a negatively charged (anionic) dye is added to a protein solution whose

pH is adjusted so that the proteins are positively charged (i.e. < the isoelectric point). The

proteins form an insoluble complex with the dye because of the electrostatic attraction

between the molecules, but the unbound dye remains soluble. The anionic dye binds to

cationic groups of the basic amino acid residues (histidine, arginine and lysine) and to free

amino terminal groups. The amount of unbound dye remaining in solution after the insoluble

protein-dye complex has been removed (e.g., by centrifugation) is determined by measuring

its absorbance. The amount of protein present in the original solution is proportional to the

amount of dye that bound to it:

Dye bound = dye initial - dye free

(a) Anionic Dye-Binding Method

Principle: The protein-containing sample is mixed with a known excess amount of anionic

dye in a buffered solution. 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.

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 (imidazole of histidine, guanidine of arginine, and -



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amino group of lysine) and the free amino terminal group of the protein . The amount of the

unbound dye is inversely related to the protein content of the sample.

Procedure

1. The sample is finely ground (60 mesh or smaller sizes) and added to an excess dye solution

with known concentration.

2. The content is vigorously shaken to equilibrate the dye binding reactions and filtered or

centrifuged to remove insoluble substances.

3. Absorbance of the unbound dye solution in the filtrate or supernatant is measured and dye

concentration is estimated from a dye standard curve.

4. A straight calibration curve can be obtained by plotting the unbound dye concentration

against total nitrogen (as determined by Kjeldhal method) of a given food covering a wide

range of protein content.

5. Protein content of the unknown sample of the same food type can be estimated from the

calibration curve or from a regression equation calculated by the least squares method.

Applications

Anionic dye binding has been used to estimate proteins in milk, wheat flour, soy

productsand meats.

. The AOAC approved methods include two dye-binding methods using Acid Orange 12

and Method using Amido Black (10B) for analyzing proteins in milk.

Advantages

1. Rapid (15 min or less), inexpensive, and relatively accurate for analyzing protein content

in food commodities.

2. May be used to estimate the changes in available lysine content of cereal products during

processing since the dye does not bind altered, unavailable lysine. Since lysine is the limiting

amino acid in cereal products, the available lysine content represents protein nutritive value

of the cereal products.

3. No corrosive reagents.

4. Does not measure nonprotein nitrogen.

5. More precise than the Kjeldahl method.

Disadvantages

1. Not sensitive; milligram quantities of protein are required.



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2. Proteins differ in basic amino acid content and so differ in dye-binding capacity.

Therefore, a calibration curve for a given food commodity is required.

3. Not suitable for hydrolyzed proteins due to binding to N-terminal amino acids.

4. Some nonprotein components bind dye (i.e., starch) or protein (calcium or phosphate) and

cause errors in final results. The problem with calcium and heavy metal ions can be

eliminated using properly buffered reagent that contains oxalic acid.

e) Turbimetric method

principle

Protein molecules which are normally soluble in solution can be made to precipitate by the

addition of certain chemicals, e.g., trichloroacetic acid. Protein precipitation causes the

solution to become turbid. Thus the concentration of protein can be determined by measuring

the degree of turbidity.

Low concentration of trichloroacetic acidand sulphosalicylic acid, potassium ferric cyanide

in aminoacid can be used to precipitate extracted proteins to form a turbid suspension.

This turbidity can be used for protein quantification.

Procedure

This method is used for the analysis of proteins by sulphosalicylic acid method.

A sample of wheat flour is extracted with 0.05 NAOH.

Protein is solubilized in alkali and is separated from the nonsoluble material by

centrifugation.

sulphosalicylic acid is mixed with a portion of a protein solution.

The degree of turbidity is measured byreading light transmittance at 540NM against the

reagent blank..

Advantages

UV-visible techniques are fairly rapid and simple to carry out, and are sensitive to low

concentrations of proteins.

Disadvantages

For most UV-visible techniques it is necessary to use dilute and transparent solutions, which

contain no contaminating substances which absorb or scatter light at the same wavelength as

the protein being analyzed. The need for transparent solutions means that most foods must

undergo significant amounts of sample preparation before they can be analyzed, e.g.,



615


homogenization, solvent extraction, centrifugation, filtration, which can be time consuming

and laborious. In addition, it is sometimes difficult to quantitatively extract proteins from

certain types of foods, especially after they have been processed so that the proteins become

aggregated or covalently bound with other substances. In addition the absorbance depends on

the type of protein analyzed (different proteins have different amino acid sequences).

4) BRADFORDMETHOD

Principle

•When Coomassie Brilliant Blue G-250 binds to protein, the dye changes color from reddish

to bluish, and the absorption maximum of the dye is shifted from 465 to 595 nm.

•The change in the absorbance at 595 nm is proportional to the protein concentration of the

sample . Like other dye-binding methods, the Bradford relies on the amphoteric nature of

proteins

• When the protein containing solution is acidified to a pH less than the isoelectric point of

the protein(s) of interest, the dye added binds electrostatically.

• Binding efficiency is enhanced by hydrophobic interaction of the dye molecule with the

polypeptide backbone adjoiningpositively charged residues in the protein .

• In the case of the Bradford method, the dye bound to protein has a change in absorbance

spectrum relative to the unbound dye.

Procedure

1. Coomassie Brilliant Blue G-250 is dissolved in 95% ethanol and acidified with 85%

phosphoric acid.

2. Samples containing proteins (1–100 μg/ml) and standard BSA solutions are mixed with the

Bradford reagent.

3. Absorbance at 595 nm is read against a reagent blank.

4. Protein concentration in the sample is estimated from the BSA standard curve.

Applications

• The Bradford method has been used successfully to determine protein content in worts and

beer products and in potato tubers

• This procedure has been improved to measure microgram quantities of proteins

• Due to its rapidity, sensitivity, and fewer interferences than the Lowry method, the

Bradford method has been used widely for the analysis of low concentrations of proteins and

enzymes in their purification and characterizations.



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Advantages

1. Rapid; reaction can be completed in 2 min

2. Reproducible

3. Sensitive; several fold more sensitive than the Lowry method

4. No interference from ammonium sulfate, polyphenols, carbohydrates such as sucrose, or

cations such as K+, Na+, and Mg+2

5. Measures protein or peptides with molecular mass approximately equal to or greater than

4000 Da

Disadvantages

1. Interfered with by both nonionic and ionic detergents, such as Triton X-100 and sodium

dodecyl sulfate. However, errors due to small amounts (0.1%) of these detergents can be

corrected using proper controls.

2. The protein–dye complex can bind to quartz cuvettes. The analyst must use glass or plastic

cuvettes.

3. Color varies with different types of proteins.The standard protein must be selected

carefully.

5) The bicinchoninic acid assaymethod(BCA assay)

• The bicinchoninic acid assay (BCA assay), also known as the Smith assay, after its

inventor, Paul K. Smith at the Pierce Chemical Company.

• Under this method proteins reduce cupric ions to cuprous ions under alkaline conditions.

• The cuprous ions complexes with apple greenish bicinchonic acid reagent to form a

purplish colour.

Procedure

• The protein solution is mixed with bicinchonic acid reagent which contains bca sodium salt,

sodium carbonate, copper sulphate amd sodium hydroxide.

• Incubate at 370c for 30 minutes or at room temperature for 2hrs

• Read the solution at 562nm against reagent blank.

Applications

• The BCAmethod has been used in protein isolation and purification. The suitability of this

procedure for measuring protein in complex food systems has not been reported.



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Advantages

1. Sensitivity is comparable to that of the lowry method;sensitivitynof the micro-bca method

is better than that of the lowry method.

2. One step mixing is easier than in the lowry method.

3. The reagent is more stable than for the lowry reagent.

4. Nonionic detergent and buffer salts donot interfere with the reaction.

5. Medium concentrations of denaturing reagents doesnot interfere.

Disadvantages

1. Color is not stable with time.the analyst need to Carefully control the time for reading

absorbance.

2. Any compound capable of reducing cu2+ to cu+ will lead to color formation.

3. Reducing sugars interfere to a greater extent than in the lowry method.high concentrattions

of ammonium sulfate also interfere.

4. Color variations among proteins are similar to those in the lowry method.[7]

OTHER INSTRUMENTAL TECHNIQUES

There are a wide variety of different instrumental methods available for determining the total

protein content of food materials. These can be divided into three different categories

according to their physicochemical principles: (i) measurement of bulk physical properties,

(ii) measurement of adsorption of radiation, and (iii) measurement of scattering of radiation.

Each instrumental methods has its own advantages and disadvantages, and range of foods to

which it can be applied.

Principles

Measurement of Bulk Physical Properties

Density: The density of a protein is greater than that of most other food components, and so

there is an increase in density of a food as its protein content increases. Thus the protein

content of foods can be determined by measuring their density.

Refractive index: The refractive index of an aqueous solution increases as the protein

concentration increases and therefore RI measurements can be used to determine the protein

content.



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Measurement of Adsorption of Radiation

UV-visible: The concentration of proteins can be determined by measuring the absorbance of

ultraviolet-visible radiation (see above).

Infrared: Infrared techniques can be used to determine the concentration of proteins in food

samples. Proteins absorb IR naturally due to characteristic vibrations (stretching and bending)

of certain chemical groups along the polypeptide backbone. Measurements of the absorbance

of radiation at certain wavelengths can thus be used to quantify the concentration of protein

in the sample. IR is particularly useful for rapid on-line analysis of protein content. It also

requires little sample preparation and is nondestructive. Its major disadvantages are its high

initial cost and the need for extensive calibration.

Nuclear Magnetic Resonance: NMR spectroscopy can be used to determine the total protein

concentration of foods. The protein content is determined by measuring the area under a peak

in an NMR chemical shift spectra that corresponds to the protein fraction.

Measurement of Scattering of Radiation

Light scattering: The concentration of protein aggregates in aqueous solution can be

determined using light scattering techniques because the turbidity of a solution is directly

proportional to the concentration of aggregates present.

Ultrasonic scattering: The concentration of protein aggregates can also be determined using

ultrasonic scattering techniques because the ultrasonic velocity and absorption of ultrasound

are related to the concentration of protein aggregates present.

Advantages and Disadvantages

A number of these instrumental methods have major advantages over the other techniques

mentioned above because they are nondestructive, require little or no sample preparation, and

measurements are rapid and precise. A major disadvantage of the techniques which rely on

measurements of the bulk physical properties of foods are that a calibration curve must be

prepared between the physical property of interest and the total protein content, and this may

depend on the type of protein present and the food matrix it is contained within. In addition,

the techniques based on measurements of bulk physicochemical properties can only be used

to analyze foods with relatively simple compositions. In a food that contains many different



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components whose concentration may vary, it is difficult to disentangle the contribution that

the protein makes to the overall measurement from that of the other components.

CONCLUSION

Proteins are extraordinarily complex molecules .Of all the molecules encountered in living

organisms proteins have the most diverse functions. So the content of proteins in food plays a

very crucial role. Presented review covers the different analytical methods for the

determination of type of protein and protein contents in food. All the reported methods are

precise, andaccurate. Further researches will provide more insight into the various methods of

analysis of proteins in the coming year.

ACKNOWLEDGEMENTS

My first salutation goes to almighty and my parents for being ever so kind and courteous to

me. i am grateful to prof.DR.SYED ABDUL AZEEZ M.PHARM.PHD, the honorable

principal of deccan school of pharmacy, Hyderabad for providing facilities to do this work

and for his constant support and encouragement.

REFERENCES

1. https://www.scribd.com.

2. U. Satyanarayana, U. Chakrapani, Text Book of Biochemistry; fourth edition, 52-53.

3. http://amrita.olabs.edu.in.

4. https://books.google.co.in.

5. https://people.umass.edu/~mcclemen/581Proteins.html.

6. https://quizlet.com/8801657/precipitation-reactions-of-proteins.

7. S. Suzanne Nielsen, Text Book of Food Analysis; Fourth Edition, 136-142.


https://www.scribd.com/

http://amrita.olabs.edu.in/

https://books.google.co.in/

https://people.umass.edu/~mcclemen/581Proteins.html

a review on general methods of analysis of proteins

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