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Physical-chemical properties of proteins; methods of its determination, precipitation reactions.

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Presentation on theme: "Physical-chemical properties of proteins; methods of its determination, precipitation reactions."— Presentation transcript:

1 Physical-chemical properties of proteins; methods of its determination, precipitation reactions

2 Separation of Amino Acids and Proteins 1.Chromatography – the method of separating amino acids on the basis of differences in absorption, ionic charges, size and solubility of molecules 2.Electrophoresis – effects separation in an electric field on the basis of differences in charges carried by amino acids and proteins under specific condition 3. Ultracentrifugation – effects separation on the basis of molecular weight when large gravitational forces are applied in the ultracentrifuge. 4. Precipitation Methods – salts as sodium sulfate, ammonium sulfate, cadmium nitrate, silver nitrate and mercuric chloride at specific conc. precipitate some proteins while others remain in solution 5. Dialysis – is for the removal of small, crystalloidal molecules from protein solution.



5 Chromatography Much of modern biochemistry depends on the use of column chromatographic methods to separate molecules. Chromatographic methods involve passing a solution (the mobile phase) through a medium (the immobile phase) that shows selective solute components. The important methods of chromatography are: 1. Ion-Exchange Chromatography 2. Antibody Affinity Chromatography 3. Gel Filtration Chromatography 4. HPLC (High Performance Liquid Chromatography)


7 Gel Filtration Chromatography


9 Precipitation of proteins


11 Proteomics Proteomics is the science of protein expression of all the proteins made by a cell Proteome pertain to all proteins being made according to the transcriptome (RNA profile). It is often visualized by a system interaction map as seen in the proteogram.

12 Procedures of the Proteomics Commonly used procedures by Proteomics are: Mass Spectrophotometry – detects exact mass of small peptides (molecular weight). X-ray Crystallography – determines 3D shape of molecules mathematically NMR Spectroscopy – magnetic signal indicates distances between atoms

13 Qualitative analysis of Proteins  Precipitation reactions  Colour Reactions of Proteins  Precipitation reactions  Precipitation by salts solution due to hydration of  Protein exist in colloidal solution due to hydration of polar groups (-COO, NH 3 +, -OH) by dehydration or  They can be precipitated by dehydration or neutralization of polar groups.  To 2 ml of protein solution add equal volume of saturated (NH 4 ) 2 SO 4 solution  White precipitation is formed

14  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

15  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 heat  Take few ml of protein solution in a test tube and heat over a flame. Cloudy white precipitation is observed  Precipitation by acids  To 1 ml of protein solution in test tube, add few drops of 1% acetic acid, white precipitation is formed 15

16 Colour Reactions of Proteins  Proteins give a number of colour reactions with different chemical reagents due to the presence of amino acid  Biuret test  The Biuret test is a chemical test used for detecting the presence of peptide bondschemical testpeptide bonds  In the presence of peptides, a copper (II) ion forms violet-colored coordination in an alkaline solutioncopperionvioletalkaline

17  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 (CuSO 4 ) solution  Formation of Purplish Violet Colour indicates the presentation of proteins 17

18 Biuret Test

19 Chemical Reaction in the Biuret test

20  Xanthoproteic Test  To 2 ml of protein solution add 1 ml conc.HNO 3  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 20 yellow

21  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(NO 3 ) 2 in nitric acid and traces of HNO 2, a red colour is produced 21

22  Ninhydrin Test  When protein is boiled with a dilute solution of ninhydrin, a violet colour is produced Proteins Hydrolysis Amino acids Amino Acids + Ninhydrin Keto acid + NH 3 + CO 2 + Hydrindantin NH 3 + Ninhydrin Pink colour 22

23  Aldehyde Test  To 1 ml of protein solution in test tube add few ml of PDAB in H 2 SO 4.  Mix the contents and heat if necessary.  The formation of purple colour is observed  Phenol’s reagent Test  To 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 23

24 Color Reactions of Proteins Test Composition of Reagent + Result (Color)Group ResponsibleImportance NinhydrinTriketohydrin HydrateBlue or Purple Free amino and free COOH Test for amino acid, peptides in determining amino acids BiuretNaOH + CuSO 4 VioletPeptide linkages + Tripeptides up to protein Millon’sHg in HNO 3 Red Hydroxyphenyl group + Tryptophan XanthoproteicConc. HNO 3 Lemon yellowBenzene ring + Tyrosine, Phenyl alanine, Tryptophan Hopkins-Cole Glyoxylic acid and conc. H 2 SO 4 Violet ringIndole group+ Tryptophan LiebermannConc. HCl, sucroseVioletIndole group+ Tryptophan Erlich’s Diazo Pb(OAc) 2 Sullfanilic acid in HCl + NH 4 OH Red orange – lighter orange + Histidine and Tyrosine Sakaguchi 10% NaOH, ά naphtol, alkaline hypobromite Intense red color Guanidine+ Arginine Acree-RosenheimHCHO conc. H 2 SO 4 Violet ringIndole group+Tryptophan Reduced SulfurKOH, Pb(OAc) 2 Black pptSulfur + Cystine, Cystein and methionine Br waterBr.H 2 O, amyl alcoholPinkIndole group+ Tryptophan Molisch ά naphtol in alcoholic H 2 SO 4 Violet ringCarbohydratesGlycoprotein AdamskiewezGlacial Acetic acid and conc. H 2 SO 4 Reddish violet ring at the junction Indole group+ Tryptophan 24

25 Quantitative Analysis of Proteins 25

26  Kjeldahl method  The 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 food 26

27  Kjeldahl method  Principles  Digestion  Neutralization  The food sample to be analyzed is weighed into a digestion flask (NH 4 ) 2 SO 4 + 2 NaOH 2NH 3 + 2H 2 O + Na 2 SO 4 H 3 BO 3 (boric acid) H+ H+ H 3 BO 3  Titration NH 4 + + H 2 BO 3 - (borate ion)

28  Enhanced Dumas method A sample of known mass Combustion (900 o C) CO 2, H 2 O and N 2 Nitrogen Thermal conductivity detector The nitrogen content is then measured

29 Methods using 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  Principles  Direct measurement at 280nm  Biuret Method  Lowry Method  Dye binding methods  Turbimetric method 29

30  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  Biuret Method A violet-purplish color is produced when cupric ions (Cu 2+ ) interact with peptide bonds under alkaline conditions The absorbance is read at 540 nm

31 31  Lowry Method  The 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

32 32  Other Instrumental Techniques  Measurement of Bulk Physical Properties  Measurement of Adsorption of Radiation  Measurement of Scattering of Radiation  Methods Based on Different Solubility Characteristics  Salting out  Isoelectric Precipitation  Solvent Fractionation  Ion Exchange Chromatography  Affinity Chromatography  Separation Due to Size Differences  Dialysis  Ultra-filtration  Size Exclusion Chromatography  Two Dimensional Electrophoresis

33 33 Amino Acid Analysis  Amino 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.

34 Name of the TestReagent UsedPositive resultsRemarks Biuret TestNaOH, dilute CuSO 4 violet+ results with polypeptides and proteins Xanthroproteic Test Conc. H 2 SO 4 AA with benzene ring (yellow) Proteins with trp, tyr, phe Million’s TestHg(NO 3 ) and Hg(NO 2 ) 2 Tyrosine (red)+ phenolic compounts Sulphur TestLead acetate, dissolved with NaOH Gray or black precipitate + lead sulphide, formed as the result of the decomposition of the cysteine by the alkali. Hopkins Cole TestGlyoxylic acid, sulfuric acid Tryptophan (violet ring) + with any compound with indole ring Ninhydrin TestninhydrinFree-NH 2 group (blue) + results given by NH 3, primary amines, amino acids, peptides, and proteins

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