Quantitative estimation of protein Likhith K

QUANTITATIVE ESTIMATION OF
PROTEIN
By
Mr. LIKHITH K
(Research Scholar)
Department of Biomedical Engineering
Manipal Institute of Technology
Eshwar Nagar, Manipal, Karnataka-
576104

CONTENTS
Introduction
Levels of protein structure
Functional characteristic
Quantitate estimations of proteins
 Lowry’ method
 Bradford method
 Biuret method
 Bicinchoninic Acid (BCA)
Conclusion
Reference

INTRODUCTION
Proteins are polypeptide structures consisting of one or more long chains of amino acid residues.
They carry out a wide variety of organism functions, including DNA replication, transporting
molecules, catalyzing metabolic reactions, and providing structural support to cells.
A protein can be identified based on each level of its structure.
Every protein at least contains a primary, secondary, and tertiary structure.
Only some proteins have a quaternary structure as well.
The primary structure is comprised of a linear chain of amino acids.
The secondary structure contains regions of amino acid chains that are stabilized by hydrogen bonds
from the polypeptide backbone.
These hydrogen bonds create alpha-helix and beta-pleated sheets of the secondary structure.
The three-dimensional shape of a protein, its tertiary structure, is determined by the interactions of side
chains from the polypeptide backbone.
The quaternary structure also influences the three-dimensional shape of the protein and is formed
through the side-chain interactions between two or more polypeptides.
Each protein at least contains a primary, secondary, and tertiary structure.
Only some proteins have a quaternary structure as well.

LEVELS OF PROTEIN STRUCTURE
Primary Structure
Secondary Structure
Tertiary Structure
Quaternary Structure

1. Primary structure
Foundationally, a protein is a chain of amino acids bound one to another via peptide bonds.
Similar to a string of beads, these strings can twist and fold into a final protein shape.
When someone eats protein, it will break down into its amino acids.
These amino acids are composed of a central carbon atom bonded to an "amino" or nitrogen-containing group
and a carboxylic "acid" group, hence the name "amino acid."
The carbon also has a single hydrogen atom and a side chain, or "R-group," which is unique to each amino acid.
The exception to this is Proline, which is a ring structure.

2. Secondary Structure
The secondary structure is the protein structure level at which two common confirmations occur,
1. alpha-helix
2. beta-pleated sheets
Biologically, this level can further subdivide into three common structures.
The third is a combination of alpha-helices and beta-plated sheets, which can form some enzymes.
The first is alpha-helices, which can be present in proteins such as hemoglobin and intermediate filaments.
Alpha helixes are amino acids in a coiled or spiral confirmation, allowing hydrogen bonding to form between
nitrogen and hydrogen, otherwise known as nitrate groups of one amino acid with the carboxyl group of another
amino acid four residues earlier. Some amino acids are more prone to form alpha helixes.
Some examples being methionine, glutamine, cysteine, histidine, and lysine.
The second is beta-pleated sheets, which appear in fatty acid transport proteins and antibodies.
Beta pleated sheets are amino acids in a series of adjacent rows, allowing for lateral hydrogen bonds to form
between the amino acid group with the carboxyl group of another amino acid.
When there is a kink in the pattern, it indicates that there is a proline amino acid located at that position.
Some amino acids are more prone to form beta-pleated sheets.
Some examples being isoleucine, tyrosine, tryptophan, and valine.

 Furthermore, there are two biological characteristics of secondary structure—those that are functional and those that are
acute phase reactants.
 The bone marrow and liver make those classified as functional.
 The bone marrow produces immunoglobulins.
 In the liver, albumin, fibrinogen, and alpha-1-antitrypsin are all produced.
 The liver produces approximately 90% of the proteins that serve as the osmotic gradient within the serum.
 An osmotic gradient is needed to pull fluid in and out of the capillaries.
 The acute phase reactants are those proteins made during inflammation.
 They are non-specific and used to monitor whether inflammation is occurring in the body.
 Examples include transferrin and ceruloplasmin.
3. Tertiary Structure
 The most critical factor that contributes to the tertiary structure is its hydrophobic and hydrophilic interactions.
 The hydrophobic fat-soluble amino acids within a protein will fold towards the protein's "inside" and away from
contact with water.
 In contrast, hydrophilic water-soluble amino acid residues will fold towards a protein's "outside," towards contact with
water.
 Also, tertiary is the level at which covalent bonds form.
 All of these characteristics contribute to the three-dimensional shape that ultimately forms.

4. Quaternary Structure
Quaternary structure is the level at which two or more proteins interact with each other and is termed
cooperativity.
When cooperativity is applied to the composition of enzymes, it classifies as an allosteric enzyme.
And allosteric enzymes are usually rate-limiting enzymes.
They are the proteins that control a specific step within a pathway.
The rate-limiting enzyme is therefore involved in the step that is termed the" rate-limiting step."
The rate-limiting enzyme should be the slowest catalyst within the reaction.
It also bears mention that most genetic conditions that are clinically significant affect the rate-limiting enzyme of
a biochemical reaction.
The kinetic curve of an enzyme will be shaped in a specific way if the protein is allosteric.
The most likely confirmation is sigmoidal, which is significant in first-order elimination.
When proteins have multiple active sites, their Vmax increases.
Proteins are said to be saturated when a substrate occupies all active sites.
Clinically this is how protein channels and transporters affect glomerular filtration rate.

Enzyme structure
Sigmoid curve of allosteric enzyme

Functional characteristics
Each amino acid consists of a carboxyl group, an amino group, and a side chain.
 Amino acids are linked together by joining the amino group of one amino acid with the carboxyl
group of the adjacent amino acid.
Each amino acid side chain has differing properties.
Some side chains can be either acidic or basic, while others can be polar uncharged or just non-polar.
These characteristics provide insight into whether the protein generally functions better in acidic or
basic environments, solubility in water or lipids, the temperature range for optimal protein function,
and which parts of the protein are found on the protein interior being in contact with the external
aqueous environment.
Some amino acids contained within the polypeptide chain can even create ionic bonds and disulfide
bridges.
The location of certain amino acids in the primary structure dictates how the secondary, tertiary, and
quaternary structure will look.

Nonpolar, Aliphatic Amino Acids - backbone molecules of the amino acid are used to form hydrogen bonds.
Glycine - can cause a bend when used in an alpha helix chain (secondary structure)
Alanine
Valine
Leucine
Isoleucine
Methionine

Nonpolar, Aromatic Amino Acids - backbone molecules of the amino acid are used to form hydrogen bonds.
Phenylalanine
Tryptophan
Tyrosine

Polar, Uncharged Amino Acids - backbone/ side chain molecules of the amino acid can be used to form hydrogen
bonds (besides proline and cysteine)
Serine
Threonine
Asparagine
Glutamine
Proline
Cysteine

Acidic Amino Acids - can be used to form hydrogen bonds (backbone/ side chain molecules) and salt bridges (side
chain molecules only)
Aspartic Acid/Aspartate
Glutamic Acid/Glutamate
Basic Amino Acids - can be used to form hydrogen bonds (backbone/ side chain molecules) and salt bridges (side
chain molecules only)
Lysine
Arginine
Histidine

Functions
Proteins serve crucial roles in human biochemistry.
The major role is to provide the body's building blocks.
They are the precursors of several biologically relevant molecules.
Therefore either the excess or deficiency of protein can lead to disease result in nervous system defects,
metabolic problems, organ failure, and even death.
Biochemical Functions
Enzymes proteins accelerate a reaction as a catalyst.
Catalyzed reactions are one million or more times faster.
Enzymes usually have the suffix "-ase" in their name.
Exceptions are enzymes discovered before the start of the naming scheme.
Each enzyme is regulated by competitive and noncompetitive inhibitors and/or by allosteric molecules.
Enzymes can catalyze pathways to produce or break down biological molecules.
Changes to enzymes can lead to disease or treatment.
Specific amino acids form an enzyme's substrate-binding site.
A substrate-binding site is the "active site."

This serves in chemical reactions.
Substrates can be hydrophobic, hydrophilic, charged, uncharged, neutral, or a combination.
Mutations that change amino acids in the active site change the enzyme's activity.
A substrate will join an enzyme that is lined with compatible amino acids.
If these amino acids change, a substrate may not be able to join, therefore rendering an enzyme non-
functional.
How a substrate interacts with an active site signifies the "affinity" of that enzyme.
Greater affinity means fewer substrate is needed to achieve a reaction.
A mutation that changes the active site can raise or lowers the affinity.
Structural Functions
Proteins serve as the structural elements of cells and tissues, the proteins actin and tubulin form actin
filaments and microtubules.
In muscle, actin provides the "scaffolding" against which myosin can produce muscle contraction.

Kinetic Functions
Motor proteins transport molecules inside a cell, provide movement of certain parts of individual cells
involved in specialized function, generate larger-scale movements of fluids and semisolids such as the
circulation of blood and movement of food through the digestive tract, and finally provide movement of
the human body through their roles in skeletal muscles.
Myosin is a protein with a hydrophobic tail, a head group that can attach and detach from actin filaments,
and a "hinge" section, which moves the head group back and forth, resulting in movement.
Channels
Channels are essential for the transportation of nutrients into and out of cells and for nerve signals and
the selective filtration of molecules in the kidneys.
This is exemplified in how the mammalian cell has an intracellular potassium concentration of
approximately 140 millimoles per cell and sodium of 5 to 15 millimoles.
The extracellular environment has a potassium concentration of 5 moles and a sodium concentration of
145 millimoles.
potassium specific channels are responsible for regulating these concentrations in their respective
compartments.

QUANTITATIVE ESTIMATION OF PROTEIN
Accurate protein quantitation is essential to protein studies in a multitude of research topics.
A wide array of different methods have been developed to quantitate both complex mixtures of proteins as well as
a single type of protein.
Total protein quantitation methods comprise traditional methods such as the measurement of UV absorbance at
280 nm, Bicinchoninic acid (BCA) and Bradford assays, as well as alternative methods like Lowry or novel
assays developed by commercial suppliers, which often provide a well-designed, convenient kit for each type of
the assay.
Individual protein quantitation methods include enzyme-linked immunosorbent assay (ELISA), western blot
analysis, and more recently, mass spectrometry, among others.
Accurate protein quantitation is essential to all experiments related to proteins studies in a multitude of research
topics.
Different wide array of different methods have been developed to quantitate both complex mixtures of proteins in
a given assay for total protein content and as well as for a single type of protein.

 ESTIMATION OF PROTEIN BY LOWRY’S METHOD
Aim: To estimate the amount of protein in the given sample by Lowry’s method.
Principle: The principle behind the Lowry method of determining protein concentrations lies in the reactivity of
the peptide nitrogen[s] with the copper [II] ions under alkaline conditions and the subsequent reduction of the Folin
Ciocalteu reagent’s phosphomolybdic and phosphotungstic acid to heteropolymolybdenum blue by the copper-
catalyzed oxidation of aromatic acid.
The Lowry method is sensitive to pH changes and therefore the pH of assay solution should be maintained at 10
- 10.5.
The Lowry method is sensitive to low concentrations of protein.
The major disadvantage of the Lowry method is the narrow pH range within which it is accurate.
However, we will be using very small volumes of sample, which will have little or no effect on pH of the
reaction mixture.

A variety of compounds will interfere with the Lowry procedure.
These include some amino acid derivatives, certain buffers, drugs, lipids, sugars, salts, nucleic acids and
sulphydryl reagents, ammonium ions, zwitter ionic buffers, nonionic buffers and thiol compounds may also
interfere with the Lowry reaction.
These substances should be removed or diluted before running Lowry assays.
Reagents
2% Na2CO3 in 0.1 N NaOH (A)
1% Sodium Potassium Tartrate in H2O (B)
0.5% CuSO4.5 H2O in H2O (C)
Reagent I: 48 ml of A, 1 ml of B, 1 ml C
Reagent II- 1 part Folin-Phenol [2 N]: 1 part water (Folin Ciocalteu reagent Water, 1:1)
BSA Standard - 1 mg/ ml

Procedure:
Take 0.2, 0.4, 0.6, 0.8 and 1 ml of BSA working standard in 5 test tubes and make up to 1ml using distilled water.
Test tube with 1 ml distilled water serve as blank.
Add 4.5 ml of Reagent I to all the tubes and incubate for 10 minutes.
After incubation add 0.5 ml of reagent II to all the tubes and incubate for 30 minutes.
Measure the absorbance at 660 nm and plot the standard graph.
Estimate the amount of protein present in the given sample with respect to concentration from the standard graph.

 ESTIMATION OF PROTEINS BY BRADFORD METHOD
Aim: To estimate the amount of protein in the given sample by Bradford Assay.
Principle : The protein in solution can be measured quantitatively by different methods. The methods described by
Bradford uses a different concept-the protein‘s capacity to bind to a dye, quantitatively. The assay is based on the
ability of proteins to bind to coomassie brilliant blue and form a complex whose extinction coefficient is much
greater than that of free dye.
Reagents:
Dissolve 100mg of Coomassie-Brilliant blue G250 in 50 ml of 95% Ethanol.
Add 100 ml of 85% phosphoric acid and make up to 600 ml with distilled water.
Filter the solution and add 100 ml of glycerol, then make upto 1000ml.
The solution can be used after 24 hrs.
BSA (1mg/ml)

Procedure:
Prepare various concentration of standard protein solutions from the stock solution (0.2, 0.4, 0.6, 0.8
and 1 ml ) into series of test tubes and make up the volume to 1 ml .
A tube with 1 ml of water serves as blank
Add 5 ml of coomassie brilliant blue to each tube and mix by vortex or inversion.
Incubate for 30 minutes and read the absorbance at 595nm.
Plot the absorbance of the standards verses their concentration.
From the standard graph find the concentration of unknown sample.

 ESTIMATION OF PROTEINS BY BIURET METHOD
Aim: To estimate the amount of protein in the given sample by Biuret Assay.
Principle: The copper (II) present in the reaction binds itself to the nitrogen atoms that are present in the protein
peptides. Since this test is not greatly disturbed by the presence of amino acids in the sample, it can be used to gauge
the concentration of proteins in whole tissue samples. However, the samples of proteins that are purified via
ammonium sulfate ((NH4)2SO4) precipitation are not ideal for this test since buffers like ammonia interfere with it.
The reaction between the copper (II) ions and the nitrogen belonging to the peptide bonds results in the
displacement of peptide hydrogens (as long as the environment is sufficiently alkaline). Now, four nitrogen atoms
donate lone pairs to form coordinate covalent bonds with the cupric ion, resulting in the formation of a chelate
complex. This chelate complex has the ability to absorb light with a wavelength of 540nm, which imparts a purple
colour to it. Therefore, the formation of a purple colored complex indicates the presence of proteins in the analyte.
Note that the concentration of peptide bonds in the analyte contribute to the intensity of the purple colour.

Biuret reagents:
Copper sulfate
Sodium hydroxide
Sodium potassium tartarate (commonly known as Rochelle salt)
Preparation Biuret reagent (1000ml)
Take 1.5 gram of pentavalent copper sulphate (CuSO4) and 6 gram of Sodium potassium tartarate and dissolve
them in 500 ml of distilled water
Note, Sodium potassium tartarate is a chelating agent and it stabilize the copper ion
Take 375 ml of 2 M Sodium hydroxide
Mix both the solution in volumetric flask and make it final volume to 1000 ml by adding distilled water.

Reagents:
Biuret reagent
Procedure:
Take two clean and dry test tubes.
Add 1-2 ml of the test solution in the respective test tubes.
Add 1-2 ml of Biuret reagent to all the test tubes.
Shake well and allow the mixtures to stand for 5 minutes.
Observe for any color change.
Result:

 ESTIMATION OF PROTEIN BY BICINCHONINIC ACID PROTEIN ASSAY (BCA)
Aim: To estimate the amount of protein in the given sample by Bicinchoninic Acid Protein Assay.
The principle of the Bicinchoninic acid (BCA) protein assay relies on the formation of a Cu2+-protein complex
under alkaline conditions, followed by reduction of the Cu2+ to Cu1+. The amount of reduction is proportional to
the amount of protein present. BCA forms a purple-blue complex with Cu1+ in alkaline environments, thus
providing a basis to monitor the reduction of alkaline Cu2+ by proteins.
This assay can be used to quantify proteins in the concentration range from 0.2 to 1.0mg/ml. It is compatible with
many detergents but not compatible with reducing agents such as dithiothreitol above 1mM. It is always advisable
to prepare the standard in the same buffer as the sample to minimize any interference effects. BCA assays are
routinely performed at 37ºC. Color development begins immediately and can be accelerated by incubation at higher
temperatures. Higher temperatures and/or longer incubation times can be used for increased sensitivity.

Reagents:
Bicinchoninic Acid Protein Assay Kit
BSA 1mg/ml
Preparation of the BCA working reagent
BCA reagents A and B are available commercially from a number of different sources.
Mix 50 parts of Reagent A (a solution containing bicinchoninic acid, sodium carbonate, sodium tartrate and sodium
bicarbonate in 0.1N NaOH, pH 11.25) with 1 part of Reagent B (4% (w/v) CuSO4.5H2O), preparing sufficient
reagent for all the standards and samples.
2ml of reagent is required for each sample.
Mix until the solution is a uniform light green colour.
The solution is stable for 1 day.

Procedure:
Prepare various concentration of standard protein solutions from the stock solution (0.2, 0.4, 0.6, 0.8 and 1 ml )
into series of test tubes and make up the volume to 1 ml .
A tube with 1 ml of water serves as blank
 Add 2 ml of the BCA reagent to each test tube, vortex gently and follow one of the following incubation
parameters:
a. 60ºC for 15 minutes
b. 37ºC for 30 minutes
c. Room temperature from two hours to overnight
Transfer the samples to cuvettes and measure the absorbance at 562nm
Plot the absorbance of the standards verses their concentration.
From graph find amount of protein concentration in unknown sample.

CONCLUSION
Proteins are bio polymeric structures composed of amino acids, of which there are 20 commons found
in biological chemistry.
Proteins serve as structural support, biochemical catalysts, hormones, enzymes, building blocks, and
initiators of cellular death.
Proteins can be further defined by their four structural levels: primary, secondary, tertiary, and
quaternary.
To know proteins in detail, protein assays are one of the most widely used methods in life science
research.
Estimation of protein concentration is necessary in biotechnology, cell and molecular biology and
other biological research applications.
It is necessary before processing protein samples for isolation, separation and analysis.
Quantitative refers to a type of information based in quantities or else quantifiable data (determination
of unknown protein concentration in a sample using standard curve obtained, (eg. All the methods
discussed above ).
Sensitivity of an assay is a measure of how little of the analyte the method can detect, Whereas
Specificity of an assay relates to how good the assay is in discriminating between the requested analyte
and interfering substances.

REFERENCE
Hanne K etal, Protein Determination-Method Matters.Foods MDPI. Jan,2018, 7, 5
Lowry OH, Rose Brough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol
Chem. 1951 Nov;193(1):265-75
Bradford, M.M.# (1976),Rapid and sensitive method for the quantitation of microgram quantities of protein
utilizing the principle of protein-dye binding",# Anal. Biochem.,#72: 248–254.

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