Definition, Classification and Chemistry of Proteins
Classification and Chemistry of Proteins
Objectives
At the end of this lecture, students will be able:
• Define proteins
• Classify proteins
• Discuss the primary, Secondary, Tertiary, and Quaternary structures of proteins
Proteins
Definition
• Organic nitrogenous substances, polymers of alpha amino acids
Classification
• Function
• Chemical nature & Solubility properties
• Nutritional importance
Proteins – Classification
Based on chemical nature and solubility properties
• Simple proteins
– Globular
– Fibrous
• Conjugated proteins
• Derived proteins
– Primary derived
– Secondary derived
Simple proteins: Composed of only amino acid residues
Globular proteins
• Albumins: Serum albumin, ovalbumin, lactalbumin
• Globulins: Serum globulins, vitelline
• Glutelins: Glutelin from wheat, oryzenin from rice
• Prolamines: Gliadin from wheat, zein from maize
• Histones: Thymus histones, histones of cod fish sperm
• Globins
• Protamines: Sperm proteins
• Albumins: Soluble in water and dilute salt solutions and coagulated by heat.
• Globulins: Soluble in neutral and dilute salt solutions
• Glutelins: Soluble in dilute acids and alkalies and mostly found in plants
• Prolamines: Soluble in 70% alcohol
• Histones: Strongly basic proteins, soluble in water and dilute acids but insoluble in dilute ammonium hydroxide
• Globins: these are generally considered along with histones, but they are not basic in nature and are not precipitated by ammonium hydroxide
• Protamines: They are strongly basic and resemble histones but smaller in size and soluble in ammonium hydroxide, They are also found in association with nucleic acids
Fibrous proteins
• Collagens: These are connective tissue proteins lacking tryptophan. Collagens, on boiling with water and dilute acids, yield gelatin which is soluble and disgestible.
• Elastins: Elastic tissues – tendons and arteries
• Keratins: Exoskeletal structures
Conjugated Proteins
Proteins + non protein part [prosthetic part or conjugating group]
Nucleoproteins: Nucleo histones, nucleo protamines
Glycoproteins: Mucin from saliva, ovomucoid from egg white
Lipoproteins: Serum lipoproteins, membrane lipoproteins
Phosphoproteins: Casein from milk, vitelline from egg yolk
Chromoproteins: Hemoglobins, cytochromes
Metallo proteins: Copper in ceruloplasmin, Zinc in Carbonic anhydrase
Derived Proteins
• Denatured or degraded products
• Primary derived proteins – denatured or coagulated or first hydrolysed products
– Coagulated proteins: heat, acids, alkalies etc. Eg. Cooked protiens, Coagulated albumin
– Proteans: earliest products of hydrolysis by enzymes, dilute acids, alkalies etc Eg. Fibrin formed from fibrinogen
– Metaproteins: Second stage products of hydrolysis with slightly stronger acids and alkalies Eg. Acid and alkali metaproteins
• Secondary derived proteins: progressive hydrolytic products
– Eg. Proteoses, peptones, polypeptides, peptides
Chemistry of Proteins
• Primary structure
• Secondary structure
• Tertiary structure
• Quaternary structure
Primary Structure of Proteins
Determination of Primary structure
• Determination of amino acid composition
• Degradation of proteins/polypeptides into smaller fragments
• Determination of amino acid sequence
Determination of amino acid composition
• Acid hydrolysis
6N HCl heated at 110oC for 20-70 hours
Acid hydrolysis destroys tryptophan and converts asparagine and glutamine into aspartate and glutamate respectively.
• Alkali hydrolysis
2 to 4N NaOH at 100oC for 5-8 hours
Destroys serine, threonine and cysteine
• Enzyme hydrolysis
Pronase
Degradation of proteins into smaller fragments
• Liberation of polypeptides
– Urea and guanidine hydrochloride-covalent bonds
– Disulphide links- performic acid
• Number of polypeptides
– Dansyl chloride – Dansyl Polypeptides – Dansyl amino acids
• Break down of polypeptides into fragments
– Enzymatic cleavage: Trypsin, Chymotrypsin, Pepsin & Elastase
– Chemical cleavage: Cyanogen bromide
Determination of amino acid sequence
• Sanger’s reagent
• Edman’s reagent
Amino acid Sequencing – Sangers Method
Amino acid Sequencing – Edmans Method
Secondary Structure of Proteins
• Alpha helix
a. It is a tightly packed coiled structure with amino acid side chains extending outward from the central axis
b. It is stabilized by extensive hydrogen bonding which is formed between H atom attached to peptide N, and O atom of peptide
C. These bonds are individually weak, but collectively they are strong enough to stabilize the helix.
a. All the peptide bonds except the first and the last in polypeptide chain gets involved in hydrogen bonding.
b. Each turn of α helix contains 3.6 amino acids, travels to a distance of 0.54 nm and the spacing between the amino acids is 0.15 nm.
c. It is the stable conformation formed with lowest energy.
d. The right hand helix is more stable than the left hand helix.
• Beta pleated sheets
Tertiary Structure of Proteins
• Three dimensional arrangement
• Hydrogen bonds, disulphide bonds, ionic interactions and hydrophobic interactions
Quaternary Structure of Proteins
• Oligomers
• Monomers, protomers or subunits
• Hydrogen bonds, ionic interactions, hydrophobic interaction
Chemical tests for Proteins
Qualitative analysis of Proteins
• Precipitation reactions
• Colour Reactions of Proteins
Precipitation reactions
Precipitation by salts
• 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
• 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 acids
• 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.
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 sulphate (CuSO4) Solution
• Formation of Purplish Violet Colour indicates the presentation of proteins
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 orange
Millon’s Test
• When Millon’s reagent is added to a protein, a white precipitation is formed, which turn brick red on heating (Hg(NO3)2 in nitric acid)
Ninhydrin Test
• When protein is boiled with a dilute solution of ninhydrin, a violet colour is produced
Hopkin- Cole’s Test
• To a few ml of protein solution in a test tube add few drops of formaldehyde solution (1:500) and 2 drops of HgSO4 (Oxidant)
• Mix thoroughly and add very gently 2-4 ml of conc.HgSO4 along the sides of the test tube
• The formation of violet coloured ring at the junction of the two layers is observed
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 1ml of NaOH solution (4% w/v) and 5 drops ofphenol’s reagent
• The formation of blue coloured solution is observed
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
• Digestion
• Neutralization
• Titration
The food sample to be analyzed is weighed into a digestion flask (NH4)2SO4 + 2 NaOH
â
2NH3 + 2H2O + Na2SO4
â H3BO3 (boric acid)
NH4+ + H2BO3- (borate ion)
â
H+ H3BO3
Enhanced Dumas method
• A sample of known mass Combustion (900oC)
• 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
• Iso electric Precipitation
• Solvent Fractionation
• Ion Exchange Chromatography
• Affinity Chromatography
• Separation Due to Size Differences
• Dialysis
• Ultra-filtration
• Size Exclusion Chromatography
• Two Dimensional Electrophoresis
Summary
• Proteins are organic nitrogenous substances, polymers of alpha amino acids
• Classified based on function, chemical nature & solubility and nutritional importance
• Based chemical nature – Simple, conjugated and derived proteins
• Structure includes primary, secondary, tertiary and quaternary
• Primary structure of protein is the linear sequence of amino acids
• Amino acid sequence can be identified by Edmans and sangers reagent
• Secondary structure refers to highly regular local sub-structures, alpha helix and beta sheets
• Tertiary structure refers to the three-dimensional structure of a single, double, or triple bonded protein molecule
• Quaternary structure is the three-dimensional structure of a multi-subunit protein and how the subunits fit together