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 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

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
The colour 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 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 + CO Hydrindantin
NH3 + Ninhydrin
Pink colour

23. 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

24. 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

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 Nitrogen
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 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.

34. 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