e-mail: Pharmaravi.2011@gmail.com 1

PROTEINS

IntroductionIntroduction

Proteins are large, complex, Proteins are large, complex, organic compounds and are organic compounds and are composed mostly of amino acids composed mostly of amino acids linked with peptide bondslinked with peptide bonds

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Proteins differ from each other according to the type, number and sequence of amino acids that make up the polypeptide backboneProteins are important constituents of foods for a number of different reasonsThey are a major source of energy, as well as containing essential amino-acidsoLysine, oTryptophan,o Methionine,

oLeucine, oIsoleucine and oValine

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•Structures of ProteinsPrimary structure

Secondary

structure

Tertiary structure

Quaternary

structure 4

Primary structure•It is the linear sequence of amino acids joined together by peptide bond.•It is simple and unfolded structure of polypeptide chains.

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Secondary structure

The primary structure of protein folds to forms secondary structure. It is 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 completes folding of the sheets and helices of a secondary structure.

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Quaternary structure refers to the way individual polypeptides combine to form complexes Quaternary structure

Quaternary structure

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Amino acid

Non-polar aliphatic R-groups

An amino acid is a small organic molecule that, as the name indicates, contains both an amino component and an acid component

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Aromatic R-groups Positively charged (= basic) R-groups

Negatively charged (= acidic) R-groups

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Qualitative analysis of ProteinsPrecipitation reactions

Colour Reactions of Proteins

Precipitation reactions

Precipitation by salts

Protein exist in colloidal solsolution ution duedue to to hydration of hydration of polar groups (-COO, NH3

+, -OH)They can be precipitated by dehydration or by dehydration or neutralization of polar groups.

To 2 ml of protein solution add equal volume of saturated (NH4)2SO4 solutionWhite precipitation is formed 10

Precipitation by heavy metal salts

To 2 ml of protein solution, add few drops of Heavy Metals (lead acetate or mercuric nitrate) solution, results in white precipitation

Precipitation by alkaloidal reagent

To a few ml of sample solution add 1-2 ml of picric acid solution. Formation of precipitation indicates the presence of proteins

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Precipitation by organic solvents

To a few ml of sample solution, add 1 ml of alcohol. Mix and keep aside for 2 min. Formation of white precipitation indicates the presence of protein

Precipitation by heatTake few ml of protein solution in a test tube and heat over a flame. Cloudy white precipitation is observed

Precipitation by acidsTo 1 ml of protein solution in test tube, add few drops of 1% acetic acid, white precipitation is formed 12

CCoolloouur r Reactions of ProteinsReactions of ProteinsProteins give a number of colour reactions with different chemical reagents due to the presence of amino acid

Biuret testThe Biuret test is a chemical test used for detecting the presence of peptide bonds In the presence of peptides, a copper (II) ion forms violet-colored coordination in an alkaline solution

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To 2 ml of protein solution in a test tube add 10%

of alkaline (NaOH) solution. Mix and add 4-5

drops of 0.5% w/v copper sulphite (CuSO4)

solution

Formation of Purplish Violet Colour indicates

the presentation of proteins 14

Xanthoproteic TestTo 2 ml of protein solution add 1 ml conc.HNO3

Heat the solution for about 2 minutes and cool under tap water

A yellow colour is obtained due to the nitration of aromatic ring

Add few drops of 40% w/v NaOH solution

The colour obtained initially changes to orange 15

yellowyellow

Millon’s Test

When Millon’s reagent is added to a protein,

a white precipitation is formed, which turn

brick red on heating

Phenols and phenolic compounds, when

mixed with Hg(NO3)2 in nitric acid and traces

of HNO2, a red colour is produced16

Ninhydrin TestNinhydrin TestWhen protein is boiled with a dilute solution of ninhydrin, a violet colour is produced

Proteins Hydrolysis Amino acids

Amino Acids + Ninhydrin

Keto acid + NH3 + CO2 + Hydrindantin

NH3 + Ninhydrin

Pink colour 17

Hopkin- Cole’s TestTo a few ml of protein solution in a test tube add few drops of formaldehyde solution (1:500) and 2 drops of HgSO4 (Oxidant)

Mix thoroughly and add very gently 2-4 ml of conc.HgSO4 along the sides of the test tube

The formation of violetviolet coloured ring at the junction of the two layers is Observed

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Aldehyde TestTo 1 ml of protein solution in test tube add few ml of PDAB in H2SO4. Mix the contents and heat if necessary.The formation of purple colour is observed

Phenol’s reagent TestTo few ml of protein solution in a test tube add 1 ml of NaOH solution (4% w/v) and 5 drops of phenol’s reagent.The formation of blue coloured solution Observed

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Color Reactions of ProteinsTest Composition of Reagent + Result (Color) Group Responsible Importance

Ninhydrin Triketohydrin Hydrate Blue or Purple Free amino and free COOH

Test for amino acid, peptides in determining amino acids

Biuret NaOH + CuSO4 Violet Peptide linkages + Tripeptides up to protein

Millon’s Hg in HNO3 Red Hydroxyphenyl group + Tryptophan

Xanthoproteic Conc. HNO3 Lemon yellow Benzene ring + Tyrosine, Phenyl alanine, Tryptophan

Hopkins-Cole Glyoxylic acid and conc. H2SO4

Violet ring Indole group + Tryptophan

Liebermann Conc. HCl , sucrose Violet Indole group + TryptophanErlich’s Diazo Pb(OAc)2 Sullfanilic acid in

HCl + NH4OH

Red orange – lighter orange

+ Histidine and Tyrosine

Sakaguchi 10% NaOH, ά naphtol, alkaline hypobromite

Intense red color Guanidine + Arginine

Acree-Rosenheim HCHO conc. H2SO4 Violet ring Indole group +Tryptophan

Reduced Sulfur KOH, Pb(OAc)2 Black ppt Sulfur + Cystine, Cystein and methionine

Br water Br.H2O, amyl alcohol Pink Indole group + Tryptophan

Molisch ά naphtol in alcoholic H2SO4

Violet ring Carbohydrates Glycoprotein

Adamskiewez Glacial Acetic acid and conc. H2SO4

Reddish violet ring at the junction

Indole group + Tryptophan

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Quantitative Analysis of Proteins

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Kjeldahl methodThe Kjeldahl method was developed in 1883

by a brewer called Johann Kjeldahl

A food is digested with a strong acid so

that it releases nitrogen which can be

determined by a suitable titration technique.

The amount of protein present is then

calculated from the nitrogen concentration of

the food22

Kjeldahl methodPrinciples

Digestion Neutralization

The food sample to be analyzed is weighed into a digestion flask 

(NH4)2SO4 + 2 NaOH

2NH3 + 2H2O + Na2SO4

H3BO3 (boric acid) 

H+ 

H3BO3

Titration

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NH4+ + H2BO3

- (borate ion)

Enhanced Dumas methodA sample of known mass

Combustion (900 oC) CO2, H2O and N2

Nitrogen

Thermal conductivity detector

The nitrogen content is then measured

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Methods using UV-visible spectroscopyThese 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

PrinciplesDirect measurement at 280nmBiuret MethodLowry MethodDye binding methodsTurbimetric method 25

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Direct measurement at 280nmTryptophan and tyrosine absorb ultraviolet light strongly at 280 nmThe 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

Biuret MethodBiuret MethodA violet-purplish color is produced when cupric ions (Cu2+) interact with peptide bonds under alkaline conditions

The absorbance is read at 540 nm

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Lowry MethodThe Lowry method combines the Biuret reagent with another reagent (the Folin-Ciocalteu 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

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Other Instrumental TechniquesMeasurement of Bulk Physical PropertiesMeasurement of Adsorption of RadiationMeasurement of Scattering of RadiationMethods Based on Different Solubility CharacteristicsSalting outIsoelectric PrecipitationSolvent FractionationIon Exchange ChromatographyAffinity ChromatographySeparation Due to Size DifferencesDialysisUltra-filtrationSize Exclusion ChromatographyTwo Dimensional Electrophoresis

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Amino Acid AnalysisAmino acid analysis is used to determine

the amino acid composition of proteins.

A protein sample is first hydrolyzed

(e.g. using a strong acid) to release the amino

acids, which are then separated using

chromatography, e.g., ion exchange, affinity

or absorption chromatography.

Fats

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Lipids can be defined as Esters of Fatty acids and are naturally occurring Lipids consist of numerous fatlike chemical compounds that are insoluble in water but soluble in organic solvents

Lipid compounds include Monoglycerides, Diglycerides, triglycerides, phosphatides, cerebrosides, sterols, terpenes, fatty alcohols, and fatty acids

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Types of fatsCnH (2n+1) CO2H

CnH(2n-1)CO2H

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ClassificationClassificationI. "Simple" Carboxylic esters

A. Fats or glycerides (esters of fatty acids with glycerol e.g. acylglycerols)

MonoglyceridesDiglyceridesTriglycerides

B. Waxes

II. Complex carboxylic esters•Glycerophospholipids•Glycoglycerolipids•Glycoglycerolipid sulfates

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III. Complex lipids (containing amides)•Sphingolipids•Glycosphingolipids

IV. Precursor and derived lipids•Acids (including phosphatidic acid and bile acids)•Alcohols (including sterols)•Bases (Sphinganines, etc.)

V. Hydrocarbons•Straight-chain•Simple branched•Polyisoprenoid

VI. Lipid vitamins and hormones

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QQualitativeualitative AnAnalysis of Fatsalysis of Fats

1.1.SolubilSolubility testity test

2.2.MicroscMicroscopicopic Properties Properties

3.3.Physical testPhysical test

4.4.Emultion formationEmultion formation

5.5.Sackowski’s testSackowski’s test

6.6.Libermann-Burchrd’s testLibermann-Burchrd’s test

7.7.Zak’s reactionZak’s reaction

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1.1.Solubility testSolubility test

Few ml of oil sample

Few drops of oil in an test tube

1-2 ml of carotene

The formation of two layers

(Insoluble)

Chloroform Benzene

Results in the soluble solution

1. Microscopic Properties

Lipids appear in white shining chombic shape

crystals

2. Physical test

•A little quantity of oil on a filter paper

Few minutes

The greasy spot penetrating the filter paper36

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4. Emultion formation4. Emultion formation

A drop of oil on a watch glass

Place carefully 2-3 drops of water over it

Oil droplet is broken into fine droplets,

indicates the process of emulsification

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5. Sackowski’s test

2 ml of Organic solution (oil) in Chloroform

2 ml of conc.H2SO43 minutes

Upper chloroform layer shows red colour and lower H2SO4 layer shows yellow colour

6. Libermann-Burchrd’s test2 ml of Organic solution (oil) in Chloroform (CHCL3) 5-6 drops of Acetic anhydride & 2

drops of conc.H2SO4

Rose colour to coloured solutionBluish Green

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7. Zak’s reaction

FeCL3 in Acetic Acid conc. H2SO4

Red coloured solution

2 ml of Organic solution in Chloroform (CHCL3)

Quantitative analysis of fats•Saponification value

•Iodine value•Hydroxyl value

•Acid value

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Saponification value

The number of milligrams of  potassium

hydroxide required to  saponify 1gm of fat

 under the conditions specified

Number of moles = Mass of oil

Relative atomic mass

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IodineIodine valuevalue

The mass of iodine in grams that is

consumed by 100 grams of a 

chemical substanceUsed to determine the amount of

unsaturation in fatty acids

The higher the iodine number, the more C=C

bonds are present in the fat

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Hydroxyl valueHydroxyl valueIt is expressed as the mass of potassium hydroxide (KOH) in milligrams equivalent to the hydroxyl content of one gram of the chemical substance

Acid valueAcid valueThe mass of potassium hydroxide (KOH) in milligrams that is required to neutralize one gram of chemical substance

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Water / Moisture DeterminationWater / Moisture Determination

Karl Fischer MethodKarl Fischer MethodThe Water Determination Test (Karl Fischer Method) is designed to determine water content in substances, utilizing the quantitative reaction of water with iodine and sulfur dioxide in the presence of a lower alcohol such as methanol and an organic base such as pyridine, as shown in the following formulae

H2O+I2+SO2 + 3 C5H5N 2(C5H5N +H) I- + C5H5N + SO3

C5H5N + SO3 + CH3OH (C5H5N +H) O- SO2 +OCH3

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1.Volumetric titrationIodine required for reaction with water is

previously dissolved in water determination

TS, and water content is determined by

measuring the amount of iodine consumed as

a result of reaction with water in a sample

Volume(ml) of TS for Water determination consumed X f (mg/ml)

Water =Weight of sample (mg)

X 100

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2. Coulometric Titration2. Coulometric TitrationFirst, iodine is produced by electrolysis of

the reagent containing iodide ion, and then,

the water content in a sample is determined

by measuring the quantity of electricity which

is required for the electrolysis (i.e., for the

production of iodine), based on the

quantitative reaction of the generated iodine

with water.

References:1)http://www.britannica.com/EBchecked/topic/479680/protein/72530/The-isolation-and-d

etermination-of-proteins#toc725312)http://csb.stanford.edu/class/public/readings/Molecular_Architecture_I_Lecture2/Voet_

and_Voet_BOOK_00_Chapter6_Protein_Structure.pdf3)http://www.biology.arizona.edu/biochemistry/problem_sets/aa/aa.html; 2003.4)http://quizlet.com/8801657/recreate_set/5)http://quizlet.com/8801729/color-reactions-of-proteins-flash-cards/6)http://people.umass.edu/~mcclemen/581Proteins.html7)http://www.sigmaaldrich.com/analytical-chromatography/analytical-reagents/amino-acid-analysis.html8)Chemical Reagents9)O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall: Protein Measurement with the Folin Phenol Reagent, J. Biol. Chem. 193 (1951) 265 - 275.10) Sargent, M.G.: Fiftyfold amplification of the Lowry protein assay. Anal. Biochem. 163 (1987) 476-481.11) Smith, P.K. et al.: Measurement of protein using bicinchoninic acid. Anal. Biochem. 150 (1985) 76-85.12) Katan MB, Mensink RP, Zock PL. Trans fatty acids and their effect on lipoproteins in humans. Annu Rev Nutr 1995; 15:473-493.13) Firestone D (May-Jun 1994). "Determination of the iodine value of oils and fats: summary of collaborative study". J AOAC Int. 77 (3): 674–6. PMID 801221914) http://www.ffcr.or.jp/zaidan/FFCRHOME.nsf//$FILE/B43.pdf 46

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