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

Separation of Amino Acids and Proteins 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 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.

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 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

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 colour obtained initially changes to orange yellow

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

Ninhydrin Test Proteins Hydrolysis Amino acids 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

Aldehyde Test Phenol’s reagent 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

Color Reactions of Proteins Composition of Reagent Test 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 Liebermann Conc. HCl , sucrose Erlich’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 +Tryptophan Reduced Sulfur KOH, Pb(OAc)2 Black ppt Sulfur + Cystine, Cystein and methionine Br water Br.H2O, amyl alcohol Pink Molisch ά naphtol in alcoholic H2SO4 Carbohydrates Glycoprotein Adamskiewez Glacial Acetic acid and conc. H2SO4 Reddish violet ring at the junction

Quantitative Analysis of Proteins

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

Kjeldahl method Principles Titration Digestion Neutralization 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 Nitrogen Combustion (900 oC) CO2, H2O and N2 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

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 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

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

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.

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