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Download 03_Physical-chemical properties of proteins
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Physical-chemical properties of proteins; methods of its determination, precipitation reactions Separation of Amino Acids and Proteins 1. 2. 3. 4. 5. Chromatography – the method of separating amino acids on the basis of differences in absorption, ionic charges, size and solubility of molecules Electrophoresis – effects separation in an electric field on the basis of differences in charges carried by amino acids and proteins under specific condition Ultracentrifugation – effects separation on the basis of molecular weight when large gravitational forces are applied in the ultracentrifuge. 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 Dialysis – is for the removal of small, crystalloidal molecules from protein solution. 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) HPLC Gel Filtration Chromatography Precipitation of proteins 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. 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 Qualitative analysis of Proteins Precipitation reactions Colour Reactions of Proteins Precipitation reactions Protein exist in colloidal solution due to hydration of polar groups (-COO, NH3+, -OH) They can be precipitated by dehydration or neutralization of polar groups. Precipitation by salts To 2 ml of protein solution add equal volume of saturated (NH4)2SO4 solution White precipitation is formed 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 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 15 formed 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 bonds In the presence of peptides, a copper (II) ion forms violet-colored coordination in an alkaline solution 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 17 Biuret Test Chemical Reaction in the Biuret test Xanthoproteic Test To 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 yellow colour obtained initially changes to 20 orange 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 produced 21 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 + NH3 + CO2 + Hydrindantin NH3 + Ninhydrin Pink colour 22 Aldehyde Test To 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 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 Color Reactions of Proteins Test Ninhydrin Composition of Reagent Triketohydrin Hydrate Blue or Purple Biuret NaOH + CuSO4 Violet Millon’s Hg in HNO3 Red Xanthoproteic Conc. HNO3 Lemon yellow Hopkins-Cole Glyoxylic acid and conc. H2SO4 Conc. HCl , sucrose Pb(OAc)2 Sullfanilic acid in HCl + NH4OH Violet ring Violet Red orange – lighter orange Indole group + Tryptophan + Histidine and Tyrosine 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 Br water Br.H2O, amyl alcohol Pink Indole group + Cystine, Cystein and methionine + Tryptophan Molisch ά naphtol in alcoholic Violet ring H2SO4 Glacial Acetic acid and Reddish violet Carbohydrates Glycoprotein Liebermann Erlich’s Diazo Sakaguchi Adamskiewez + Result (Color) Group Responsible Importance Free amino and free Test for amino acid, COOH peptides in determining amino acids Peptide linkages + Tripeptides up to protein Hydroxyphenyl + Tryptophan group Benzene ring + Tyrosine, Phenyl alanine, Tryptophan Indole group + Tryptophan 24 Indole group + Tryptophan Quantitative Analysis of Proteins 25 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 Kjeldahl method Principles Digestion Neutralization Titration The food sample to be analyzed is weighed into a digestion flask (NH4)2SO4 + 2 NaOH 2NH3 + 2H2O + Na2SO4 H3BO3 (boric acid) NH4+ + H2BO3- (borate ion) H+ H3BO3 Enhanced Dumas method A sample of known mass CO2, H2O and N2 Combustion (900 oC) Nitrogen Thermal conductivity detector The nitrogen content is then measured 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 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 (Cu2+) interact with peptide bonds under alkaline conditions The absorbance is read at 540 nm Lowry Method The Lowry method combines the Biuret reagent with another reagent (the FolinCiocalteu 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 31 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 32 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. 33 Name of the Test Reagent Used Positive results Remarks Biuret Test NaOH, dilute CuSO4 violet + results with polypeptides and proteins Xanthroproteic Test Conc. H2SO4 AA with benzene ring (yellow) Proteins with trp, tyr, phe Million’s Test Hg(NO3) and Hg(NO2)2 Tyrosine (red) + phenolic compounts Sulphur Test Lead 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 Test Glyoxylic acid, sulfuric acid Tryptophan (violet ring) + with any compound with indole ring Ninhydrin Test ninhydrin Free-NH2 group (blue) + results given by NH3, primary amines, amino acids, peptides, and proteins