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

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

2. 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.

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)

6. HPLC

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 • 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

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 whiteprecipitation is observed • Precipitationbyacids • To 1 ml of protein solution in test tube, add few drops of 1% acetic acid, white precipitation isformed

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 bonds • In the presence of peptides, a copper (II) ion forms violet-colored coordination in an alkaline solution

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 (CuSO4) solution • Formation of Purplish Violet Colour indicates the presentation of proteins

18. Biuret Test

19. Chemical Reaction in the Biuret test

20. 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 • Thecolour obtained initially changes to orange 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(NO3)2 in nitric acid and traces of HNO2, a red colour is produced

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 + NH3 + CO2 + Hydrindantin NH3 + Ninhydrin Pink colour

23. 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 colourisobserved • 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 solutionObserved

25. Quantitative Analysis of Proteins

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

27. 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

28. Enhanced Dumas method A sample of known mass Combustion (900 oC) CO2, H2O and N2 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

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 (Cu2+) interact with peptide bonds under alkaline conditions The absorbance is read at 540 nm

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. 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. 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.