Protein Chemistry PDF

Protein Chemistry By Dr Randa Elbataier  Introduction - There are 4 Macromolecules in the Human Body: - Protein, Lipid, Carbohydrate, Nucleic acids. - Proteins are the Most Abundant & Most Functionally Diverse Macromolecule The Most Abundant: They Occur in all...

Protein Chemistry By Dr Randa Elbataier  Introduction - There are 4 Macromolecules in the Human Body: - Protein, Lipid, Carbohydrate, Nucleic acids. - Proteins are the Most Abundant & Most Functionally Diverse Macromolecule The Most Abundant: They Occur in all cells & in all parts of cells  The Most Functionally Diverse: They have many functions. - There are More than 100 000 Human Proteins. - There are 3000 to 5000 Proteins Within One Cell. - There are More than 1400 Proteins in Blood (Serum). - ½ of Body Lean Weight is Protein.  Introduction to Proteins Definition: - Protein are Macromolecules that Consists of a Chain of Amino Acids Connected by Peptide Bonds. - Proteins are Linear Polymers of Amino Acids. Atoms: - Proteins Consist of: - Carbons (C), Oxygen (O), Nitrogen (N), Hydrogen (H). - Sometimes Sulfur (S).  Amino Acids - Amino acids are the Basic Structural Units (Monomers) of Proteins. - They are Organic Compounds that Contain Both an Amino group (NH2) & a Carboxyl group (COOH). Structure: - Amino acids are Organic, so they Contain a Carbon. - The Second Carbon after Carboxyl carbon is α-Carbon. - The α-Carbon is Attached to: (1) Carboxyl group (COOH) (2) Amino group (NH2) (3) Hydrogen atom (H) (4) Side chain or radical group (R)  Optical Activity & Stereoisomers All are: 1. α-Amino acids: i.e. the amino group attached to the second carbon (next to the carboxyl group). 2. L-Amino acid: i.e. α-amino group is on the left side configuration.  Optical Activity & Stereoisomers -All amino acids have optical isomers except glycine. - All Amino acids have Stereoisomerism Except Glycine. - Stereoisomerism Means amino acids can be L-isomers or D-isomers. - Amino acids in Mammalian Proteins are L-Amino acids (Configuration). - L-Amino acids have the α-Amino group (NH2) on the Left Side of the α- Carbon.  Amino acids in Nature & Mammals: - Amino acids in Nature are Abundant. - There is hundreds (about 300) in nature. - But only 20 amino acids make mammalian proteins. - These are known as proteinogenic amino acids. - They are amino acids that are Coded for by DNA. - They are amino acids that are Translated into Protein. The 20 Proteogenic Amino Acids (1) Glycine, (2) Alanine, (3) Leucine, (4) Isoleucine, (5) Valine, (6) Phenylalanine, (7) Tryptophan, (8) Methionine, (9) Proline, (10) Serine, (11) Threonine, (12) Tyrosine, (13) Cysteine, (14) Glutamate, (15) Aspartate, (16) Glutamine, (17) Asparagine, (18) Histidine, (19) Lysine, (20) Arginine.  Classification of amino acids: 1- Chemical 2- Nutritional 3- Metabolic Classification Classification classification (Chemistry of the Side (Needed in Diet or Not) Their break down gives: chain) -Essential (Ketone bodies or gives (Polarity of side chain R -Semi-essential Glucose, or Both) group) -Nonessential -Pure Ketogenic (Ionization of side chain R -Pure Glucogenic group) -Mixed Glucogenic & -Non-polar -Polar Ketogenic  Chemical classification: 1. According to the fatty acids the amino acids are derived from: 1. According to the fatty acids the amino acids are derived from 1. According to the fatty acids the amino acids are derived from 1. According to the fatty acids the amino acids are derived from 1. According to the fatty acids the amino acids are derived from 1. According to the fatty acids the amino acids are derived from  Chemical classification 2. According to if the amino acid is acidic, basic or neutral amino acids: a) Acidic amino acids: contain more than one -COOH group. e.g. Aspartate and glutamate. b) Basic amino acids: contain more than one -NH2 group. e.g. lysine, Arginine and histidine. c) Neutral amino acids: these are amino acids, which contain one COOH and one -NH2 groups. e.g. glycine, alanine, etc.  Chemical classification 3. According to the polarity of the radical (R): Polar: Neutral amino acids Acidic amino acids Bacic amino acids Non polar: Rest of amino AA  Chemical classification 4. Classification according to if amino acid is: aromatic, heterocyclic or aliphatic. 1. Aromatic amino acids: which contain phenyl or phenol ring: a) Phenylalanine (phenyl ring). b) Tyrosine (phenol ring). 2. Heterocyclic amino acids: which contain other type of rings: a) Tryptophan (indole ring). b) Histidine (imidazol ring). c) Proline (pyrrolidine ring). d) Hydroxyproline (hydroxy pyrrolidine ring). 3. Aliphatic amino acids: include other amino acids which contain no ring  Chemical classification 5. According to if the amino acid is: branched or non branched: 1. Branched amino acids: valine , Leucine and lsoleucine. 2. Non-branched amino acids: Rest of amino acids. 6. Imino and amino acids: 1. Imino acids: Proline and, hydroxyproline. 2. Amino acids: Rest of amino acids. 7. Sulfur and hydroxyl containing amino acids: 1. Sulfer containing amino acids: cysteine, cystine and methionine. 2. Hydroxyl containing amino acids: serine, threonine.  Nutritional Classification  Essential Amino Acids: (8) Must be supplied from diet. can't be synthesised in body require large number of enzymes (about 59 enzymes) their deficiency affects growth & health: Methionine – Phenylalanine – Lysine – Threonine – Valine - Leucine Isoleucine – Tryptophan – Histidine.  Semi-Essential Amino Acids: (2) Synthesised in sufficient amount in healthy state. they become insufficient during pregnancy, childhood, illness & fast cellular growth: Histidine – Arginine.  Nutritional Classification Non-Essential Amino Acids: (10) Don’t have to be Supplied from diet. Can be Synthesised in Body Require Small Number of Enzymes: Glycine – Alanine – Tyrosine – Cysteine – Proline - Aspartate - Asparagine – Serine – Glutamate – Arginine.  Metabolic classification: - Amino acids are Classified according to their Metabolic fate in body. - Amino acids are Classified according to Degradation Product (Carbon Skeleton). Into Pure ketogenic amino acids: Its breakdown ONLY Gives Ketone bodies: Leucine & Lysine. Mixed ketogenic & Glucogenic Amino acids: Its breakdown Gives BOTH Ketone bodies & Glucose: Isoleucine Tryptophan Tyrosine and Phenylalanine. Pure Glucogenic amino acids: Its breakdown ONLY Gives Glucose The Remaining 14 amino acid.  Ionization of Amino acids Ionisable groups of Amino acids: - All amino acids contain 2 Ionisable groups Except Acidic & Basic Amino acids. - Acidic & Basic amino acids have 3 Ionisable groups. - These ionisable groups are the (COOH & NH2+) - Carboxyl (COOH) is ionized by losing a H+ (Deprotonation) to form Negative (Coo-) - Amino (NH2) is ionized by gained H+ (Protonation) to form Positive (NH3+) Function (biomedical importance) of amino acids A. Structural function: enter in the structure of:  Body peptides and proteins: e.g. plasma proteins, tissue proteins, enzymes, etc.  Hormone: some hormones are amino acid derivatives e.g. thyroxin.  Amines: Some amino acid gives corresponding amines by decarboxylation e.g. histidine gives histamine which is vasodilator. B. Neurotransmitters: Some amino acids as glycine and glutamate act as N.T C. Detoxication: Some amino acids are used in detoxication reactions  Peptides Definition: Peptides are compounds, formed of less than 50 amino acids Linked together by peptide bonds.  Dipeptide (2 amino acids and 1 peptide bond).  Tripeptide (3 amino acids and 2 p.b).  Oligopeptide (3-10 amino acids).  Polypeptide (10-50 amino acids). Peptide bond: - Definition: It is a covalent bond formed between the carboxyl group of one amino acid and the amino group of another. - Mechanism: - it is formed by removal of water. - Peptide formation needs energy getting from hydrolysis of ATP - Characters: - Peptide bond is semi-rigid bond i.e. no free rotation can occur around bond axis.  Primary structure of peptides: It’s the arrangement of amino acids in a polypeptide chain. - In a polypeptide chain the N-terminal amino acid (i.e. the only amino acid that contains free amino group) is always to the left side. - The C-terminal amino acid (i.e. the only amino acid that contains free carboxyl group) is aIways to the right.  BioIogicaIIy active peptide Peptides include many active compounds as: Glutathione. - Definition: it is a Tripeptide formed of three amino acids: glutamate cysteine and glycine. It is also called “glutamyl-cysteinyl-glycine”. Glutathione is commonly abbreviated as G-SH where -SH indicates the sulfhydryl group of cysteine and it is the most active part of the molecule.  BioIogicaIIy active peptide - Functions of glutathione: 1- Defense mechanism against certain toxic compounds (Detoxification). 2-Absorption of amino acid: glutathione has a role in transport of amino acids across intestinal cell membrane.. 3- Protect against cell damage and hemolysis of RBCs: Glutathione breakdown the hydrogen peroxide (H2O2) which causes cell damage and hemolysis. 4- Activation of some enzymes. 5- Inactivation of insulin hormone.  Proteins Nature of proteins:  Composition: 1. Proteins are macromolecules formed of amino acids united together by peptide bonds. 2. Amino acids are commonly found in proteins in different proportions. 3. Some proteins are formed of 2 or more polypeptide chains.  Size of proteins: 1. Proteins having a very high molecular weight, ranging from 5,000 to several millions. 2. The term protein is applied to describe molecules greater than 50 a.a. 3. Molecules contain less than 50 amino acids are termed peptides.  Functions of proteins: 1. Enzymes: Enzymes are protein 2. Transport: Of small molecules and ions e.g. a. Hemoglobin is a carrier for oxygen. b. Lipids are transported as lipoproteins. 3. Structural elements: e.g. a. Cell membrane contains proteins in the form of glycoproteins. b. Skin and bone: e.g. contains proteins in the form of collagen. 4. Hormonal regulation: a. Some hormones are protein in nature e.g. growth hormone. b. Cellular receptors that recognize hormones are proteins  Functions of proteins: 5. Defense mechanism: a. Antibodies: (immunoglobulins) are protein in nature. b. Keratin found in skin and other tissues is protein that protect against mechanical and chemical injury. 6. Blood clotting: Coagulation factors are proteins. 7. Storage: as ferritin which is a storage form of iron. 8. Control of genetic expression: many regulators of genes are protein in nature. Conformation of proteins = (protein structure): - The structure of protein is described in four different levels include: primary structure, secondary structure, tertiary structure and Quaternary Structure. Primary structure: - Definition: It is the arrangement of amino acids in the polypeptide chain. - Bonds responsible for the primary structure: The peptide bonds “covalent”.  Primary Structure Mechanism: 1. Each polypeptide chain starts on the left side by free amino group of the first amino acid, It is termed N-terminal (or N-terminus) amino acid. 2. Each.polypeptide chain ends on the right side by free carboxyl group last amino acid, It is termed C-Terminal (or C-terminus) amino acid. 3. The remaining amino acids in the chains are termed: amino acid residues 4. The types and arrangement of amino acid in each protein is determined by the genetic information present in DNA.  Secondary structure: - Definition: It is the spatial relationship of adjacent amino acid residues. - Bonds responsible: Hydrogen bond. It is the bond between the hydrogen of -NH group of one amino acid residues and the carbonyl oxygen (C=O) of the fourth one - Mechanism: 1. Secondary structure results from interaction of adjacent amino acid residues (first and fourth). 2. There are 2 main forms of secondary structure α-helix and β-pleated sheets.  Secondary structure: The α-helix β-pleated sheets - Shape & formation: It is a rod like - Shape & formation: structure with the peptide bonds coiled a) This structure is formed between two or tightly inside and the side chains of the more separate polypeptide chains. It may also residues (R) extending outward from the be formed between segments of the same chain. polypeptide chain. - Characteristics: b) Hydrogen bond is also responsible for its 1) Each (C=O) of one amino acid is formation. It occurs between (-NH) group of hydrogen bonded to the (-NH) of the next one chain (or segment) and (C=O) of group of fourth amino acid in the chain adjacent chain (or segment). (1→4 ) - Two types of β-sheets are present: 2) The complete turn distance equals 54nm. 1) Parallel β-sheets: in which the two 3) Each turn contains 3.6 amino acids polypeptide chains run in the same direction. residues. 2) Antiparallel β-sheets: in which the two polypeptide chains run in opposite direction. Secondary structure:  Tertiary structure Definition: This is the final arrangement of a single polypeptide chain resulting from spatial relationship of more distant amino acid residues. - There are two forms of tertiary structures: 1. Fibrous: which is an extended form e.g. keratin, collagen and elastin. 2. Globular: which is a compact form and results from folding of polypeptide chain e.g. myoglobin. - Bonds responsible for tertiary structure are: 1. Hydrogen bonds: within the chain or between chains 2. Hydrophobic bonds: between the nonpolar side chains (R) of neutral amino acids. 3. Electrostatic bonds: (salt bonds): between oppositely charged groups in the side chains of amino acids e.g. amino group of lysine and carboxyl group of Aspartate. 4. Disulfide bonds: between residues within the chain.  Quaternary structure - Many proteins are composed of several polypeptide chains. Each poly peptide chain is called: subunits. Each subunit has its own primary, secondary and tertiary structure, - Bonds responsible for quaternary structure: 1. Hydrogen bond. 2. Hydrophobic bond. 3. Electrostatic bond - Examples of proteins having quaternary structure: a) Insulin: 2 subunits. b) Lactate dehydrogenase enzyme: 4 subunits. c) Globin of hemoglobin: 4 subunits.  Denaturation Definition: unfolding and loss of secondary tertiary and quaternary structure. - Does not affect primary structure i.e. not accompanied by hydrolysis of peptide bond. - Denaturation may be reversible (in rare cases) Effect of protein denaturation: 1. Loss of biological activity: e.g. insulin loses its activity after denaturation. 2. Denaturated protein are often insoluble. 3. Denaturated protein are easily precipitated. Denaturating factors include: 1. Heat: causes coagulation and precipitation of certain proteins like albumin. 2. Organic solvents: They interfere with hydrophobic bonds of proteins. 3. Detergents: They contain both hydrophobic and hydrophilic groups i.e. amphipathic. They interfere with hydrophobic bonds of proteins. 4. Strong acids or bases: They lead to change in pH which affects the charges on polypeptide chains. As a result, hydrogen and electrostatic bonds will be disrupted. 5. Heavy metals: as lead and mercury salts: a) They form ionic bonds with negatively charged ions in polypeptide chains. This leads to disruption of electrostatic bonds. b) They unite with -SH (sulfhydryl) groups of proteins causing its denaturation (- S-Hg). 6. Enzymes: e.g. Digestive enzymes. 7. Urea, ammonium sulphate and sodium chloride: cause precipitation of proteins. 8. Repeated freezing and thawing: cause disruption of hydrogen and other bonds.  Classification of proteins A- Albumin and A- phosphoprotein A-Primary derived: globulins: B- Lipoproteins = Denaturated B-Basic proteins C- Glycoproteins proteins 1- Globins (histones) D- Metaloproteins B-Secondary derived: 2- Protamins E- Chromoproteins = Hydrolytic C-Acidic proteins: F- Nucleoproteins products 1- Gliadins 2- Glutelins D-Scleroproteins: 1- Keratins 2- Collagen 3-El`astin  Simple proteins A. Albumin and globulins: Albumin globulins coagulated by heat coagulable same biological value high same Solubility Soluble in water Soluble in salt solution Molecular weight 68.000 150,000 Precipitation By full saturated By half saturated ammonium sulphate ammonium sulphate Sources: Serum albumin Serum globulins 1)Blood Lactalbumin Lactglobulin 2)Milk Egg albumin Egg globulin 3)Egg  Simple proteins B. Basic proteins: Globins (=histones) and protamines: Both are basic proteins i.e. rich in basic amino acids. Globins (=histones) Protamins Type of basic amino Histidine Lysine and Arginine acid Solubility In salt solution 1. In salt solution 2. In 70% ethanol Sources: 1. In plants & In fish 1. Combined with animals DNA 2. To form 2. Combined with hemoglobin Heme  Simple proteins C. Gliadins and Glutelins: 1. Both are acidic proteins i.e. rich in acidic amino acids: glutamic acid. 2. Both are present in cereals 3. Both are soluble in diluted acids and alkalies. Gliadins also soluble in 70% ethanol. D. Scleroproteins: They include: keratin, collagen, and elastin. 1. Keratins: a) Location: They are found in hair, nail, enamel of teeth, and outer layer of skin. b) Structure: They are α-helical polypeptide chains. They are rich in cysteine  Simple proteins Collagen 2. Collagen a) Types of collagens: - There are more than 12 types of collagen. Type I is the most common in human body 90% of cell collagens. - Collagens form about 30% of total body proteins. b) Functions and Location: - It is the protein of connective tissue present in skin, bones, tendons and blood vessels. - Bones and teeth are made by adding mineral crystals to the collagen. - Collagen may be present as a gel e.g. in extracellular matrix or in vitreous humour of the eye.  Simple proteins Collagen c) Structure: 1) Collagen molecules are simple protein; consist of 3 polypeptide chains called α-chains. They are twisted around each other forming triple helix molecule. i- The 3 polypeptide chains are held together by hydrogen bonds ii- each chains about 300 nm length and 1.5nm in diameter. iii- Each chain is formed of 1050 amino acids.  Simple proteins Collagen 2) Amino acids composition acid sequence: i- Amino acids composition: Collagen contains 33% glycine (the smallest amino acid), 10% proline, 10% hydroxy proline and 1% hydroxylysine. ii- Amino acids sequence: Every third amino acid in the α-chain is glycine. The repeating sequence is glycine-X-Y, where X is frequently proline and Y is often hydroxy-proline or hydroxylysine 3) Glycosylation: Collagens are present in the form of glycoprotein. Glucose and galactose are commonly attached to collagen  Simple proteins Collagen 4) Collagen molecule has very firm structure due to: i- Each helical turn contains only 3 amino acids. For other proteins, each turn contains 3.6 amino acids. ii- Glycine (the smallest amino acid) forms 33% of total molecule. This makes the polypeptide chains compact. iii- The high content of hydroxyproline and hydroxylysine increase the number of hydrogen bonds Collagens are formed by connective tissue cells called fibroblasts. Collagen diseases: (Scurvy): It is due to a deficiency in ascorbic acid (vitamin C) Collagen synthesis: 1. Collagens are formed by connective tissue cells called fibroblasts. 2. Intracellular location: The polypeptide chains of preprocollagen are synthesized on the rough endoplasmic reticulum, where preprocollagen is cleaved → Procollagen + Signal (pre) sequence. 3. Proline and lysine residues are hydroxylated by a reaction that requires O2 and vitamin C  Simple proteins Elastin 3. Elastin: a) Characters: - It is connective tissue protein. It is rubber like i.e. it can be stretched to several times as their normal length, but recoil to their original shape when the stretching force is relaxed b) Location: It is present in lungs, the walls of large blood vessels and elastic ligaments.  Simple proteins Elastin C) Structure: 1) Elastin is formed of 4 polypeptide chain. 2) Elastin is similar to collagen, being rich in glycine (1/3 of its a.a) and proline. It is poor in hydroxyproline hydroxylysine. 3) The 4 polypeptide chains are interconnected through their lysine residues. The 4 lysine residues are linked together form a cyclic structure termed: desmosine. Elastin is capable of undergoing 2 way stretch, due to its content of desmosine.  Conjugated proteins Conjugated proteins: On hydrolysis, they give protein (prosthetic group).They include: part (apoprotein) and nonprotein part. A. Phosphoprotein: 1. These are proteins conjugated with phosphate group. 2. Phosphate is attached to -OH group of serine (phospho-serine) or threonine (phospho-threonine) present in protein part. Examples: a) Casein: A milk proteins b) VitelIin: Present in egg yolk. c) Phosphoenzyme: Phosphorylation (addition of phosphate to an enzyme) may activate or inactivate enzyme according to its type.  Conjugated proteins B. lipoproteins C. Glycoproteins and proteogIycans D. Nucleoproteins E. Chromoproteins:They are proteins conjugated with colored elements.  Metalochromoproteins (contain colored metal) 1- All iron containing proteins (red) 2-All copper containing proteins (greenish blue).  Non-metalochromoproteins (contain colored pigment) 1- Flavoprotein (yellow) contain Flavin pigment e.g. FAD. 2- Carotenoids: they give vitamin A. 3- Melanoproteins: (brown to black) e.g. melanin pigments of hair and iris.  Conjugated proteins F. Metaloproteins: These are proteins conjugated with metals. Metalloproteins containing Non-Heme iron: Ferritin, Transferrin, Hemosiderin, Lactoferrin. Metalloproteins containing Heme Iron: Hemoglobin & Cytochromes Metalloproteins containing Copper: Super Oxide Dismutase (SOD) Metalloproteins containing Zinc: Insulin, Alcohol dehydrogenase Metalloproteins containing Magnesium: Phosphatase Metalloproteins containing Manganese: Carboxylase  Conjugated proteins G. Hemoproteins are conjugated protein formed of protein part (globin) and non protein prosthetic part (Heme). 1. Hems containing iron (red in color). Thus hemoproteins are considered Metaloproteins. 2. Hemoproteins include many biologically active compounds as: a) Hemoglobin: This carries oxygen. b) Myoglobin: This stores oxygen in muscles. c) Respiratory enzymes: These use oxygen.  Conjugated proteins Structure of Heme: 1. Four Payrol rings are united together to form protoporphyrin III. 2. Iron in ferrous state (Fe++) is incorporated in protoporphyrin III to form heme.  Conjugated proteins Hemoglobin: 1. It is a Metaloproteins formed of heme and globin. Found in RBCs - Globin is a globular protein rich in histidine amino acids. It forms about of 95% of haemoglobin molecule. - Globin is a protein having a quaternary structure. It is formed of 2 α chain (each 141 amino acids) and 2 β chains (each 146 amino acids). Functions of hemoglobin: 1. Carries O2 to tissues and removes CO2 from them to the lungs. 2. Acts as blood buffer. 3. Synthesis of heme. 4. Structure of hemoglobin. 5. Properties of hemoglobin. 6. Hemoglobin derivatives.  Conjugated proteins Myoglobin 1. It is found only in the cytosol of red skeletal muscle and cardiac muscle. It gives these tissues their characteristic red color. 2. It is formed of one heme molecule attached to one polypeptide chain. This polypeptide chain contains 153 amino acid sand terminated by Proline. 80% of Myoglobin polypeptide chain is α- helix. 3. Myoglobin has much higher affinity for oxygen than hemoglobin. It is unable to release it except under very low oxygen tension. 4. Myoglobin concentration is increased in blood in myocardial infarction. The Globular protein in Myoglobin & Hemoglobin Myoglobin Hemoglobin  Clinical aspects 1. Myoglobinuria a) It is the release of myoglobin from muscles after massive crush injury. b) Myoglobin is excreted in urine, colors it dark. It may cause renal tubular obstruction and renal failure. 2. Plasma myoglobin is increased following myocardial infarction, but measurement of serum myocardial enzymes provides a more Sensitive index of myocardial infarction.  Clinical aspects: 3. Sickle cell anemia: a) The blood cells of these patients contain abnormal hemoglobin called hemoglobin S (HbS). b) A molecule of HbS contains 2 normal α-chains and 2 mutants - chains in which glutamate at position six has been replaced by valine. 4. Thalassemias: Are anemias characterized by reduced synthesis of either alpha chain (α-Thalassemias) or beta chain (β-Thalassemias) of hemoglobin.  Important Information: - P50: Means the Partial Pressure of O2 required to Saturate 50% of binding site. - The lower the P50 the Higher the affinity & the Lower the release. - Cooperativity: Means binding of one oxygen molecule enhances the binding of the other oxygen molecules. - In Hb the affinity for the last O2 is 300 times greater than the affinity for the first O2.  Oxygen Binding of Myoglobin & Hemoglobin Myoglobin Hemoglobin Oxygen molecule carrying Carries 1 oxygen molecule Carries 4 Oxygen molecules capacity Affinity to Oxygen molecules Has Higher affinity to O2 Has Lower affinity to O2 P50 1 mmHg 26 mmHg Cooperativity No cooperativity Shows cooperativity Forms No forms Has Tense & Relaxed forms Allostertic affect No Allostertic affect Allostercally affected Saturation Curve Hyperbolic curve Sigmoid curve  3-Derived proteins These are not naturally occurring proteins and are obtained from simple proteins by the action of enzymes and chemical agents. protein peptone polypeptide short peptide amino acids There are two classes of derived proteins. Primary derived proteins: These are derivatives of protein in which the size of protein molecules not altered materially Example: Coagulated proteins like cooked-egg albumin. Secondary derived proteins: These are derivatives of proteins in which the hydrolysis has certainly occurred, The molecules are smaller than the original proteins Example: Prolonged hydrolysis of natural proteins yields peptides  Techniques for Separation and purification of proteins and amino acais The basic aim in protein purification is to isolate one particular protein of interest from other contaminating proteins to study its structure and function, increasing its stability and large scale Purification of proteins is an essential first step in understanding their function Proteins must be released from the cell to be purified. Based on the basic properties of proteins like solubility, size, charge and binding affinity.  1. Electrophoresis Electrophoresis is based on the ability of particles possessing electric charge, including proteins, to migrate in continuous electric field.  1. Electrophoresis is a technique used to separate different elements (fractions) of a blood sample into individual components. Serum protein electrophoresis is a test that measures the major blood proteins by separating them into five distinct fractions: albumin, alpha1, alpha2, beta, and gamma proteins by applied to a strip of filter paper or cellulose acetate.  1. Electrophoresis When the current passes, proteins will migrate towards positive electrode (anode). The rate of migration depends on: a) The amount of charges carried by each protein. b) The molecular weight of proteins.  2. Salt precipitation it is a method of precipitating proteins from the solution under the action of neutral salts in high concentrations (ammonium sulfate, etc.) it is a reversible process the protein doesn’t lose the activity. The mechanism of salt precipitation: breakdown of the hydration shell removing the electric charge. At very high ionic strengths, the salt withdraws the hydration shall from the proteins and thus leads to aggregation and precipitation of the molecules (salting out). For this reason, adding salts such as ammonium sulfate (NH 4)2 SO4 makes it possible to separate proteins from a mixture according to their degree of solubility (fractionation).  2. Salt precipitation - The rate of precipitation of proteins by salt precipitation depends on a number of factors such as hydrophilic properties of the protein, its molecular weight, electric charge; thus the salt precipitation of various proteins occurs in various salt concentrations. - For example, albumin is precipitated in a saturated solution of ammonium sulphate, whereas globulins - the half-saturated  2. Salt precipitation -The solubility of proteins is strongly dependent on the salt concentration (ionic strength) of the medium. - Proteins are usually poorly soluble in pure water. Their solubility increases as the ionic strength increases, because more and more of the well- hydrated anorganic ions are bound to the protein’s surface, preventing aggregation of the molecules (salting in).  3. Ultracentrifugation By using a centrifuge of about 40000 rounds per minute (RPM).By this method, a mixture of proteins is separated into different fractions according to their densities.  4. Dialysis- – is the process of separating molecules in solution by the difference in their rates of diffusion through a semipermeable Membrane - method is used for macromolecular compounds (proteins) separation and purification of micromolecular compounds with the help of semipermeable membrane (cellophane, parchment, etc.). - through the pores of this membrane can pass only micromolecular compounds that have low molecular weight and small size.  4. Dialysis- Due to their size, protein molecules are unable to pass through the pores of a semipermeable membrane, while lower-molecular substances are able. Thus, dialysis can be used to remove lower-molecular components from protein solutions.  5. Chromatography Chromatography is a group of separation techniques, where a mixture of molecules is separated into its components. Types of chromatography: 1. Paper chromatography 2. Thin layer chromatography(TLC) 3. lon exchange chromatography