Proteins,Fats determination
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e-mail: [email protected] 1
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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
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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
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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
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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
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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
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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
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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
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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
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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)
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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.
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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
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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.
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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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