Amino Acids & Proteins PDF

All cells contain many types of proteins, and amino acids are the building blocks of these proteins. Proteins are of paramount importance in biological systems. All the major structural and functional aspects of the body are carried out by protein molecules. All proteins are polymers of amino acids....

All cells contain many types of proteins, and amino acids are the building blocks of these proteins. Proteins are of paramount importance in biological systems. All the major structural and functional aspects of the body are carried out by protein molecules. All proteins are polymers of amino acids. Proteins are composed of a number of amino acids linked by peptide bonds. **Amino acids** are bi-functional compounds containing both a carboxylic acid group (-COOH) and a basic amino group (-NH2) attached to the same carbon atom. The biologically important amino acids are the α-amino acids that have the amine and acid groups attached to the same carbon atom. There are more than 300 known natural amino acids; however, only 20 are used in protein synthesis. **General Properties of Amino Acids** Like any organic compound, the properties of the molecules are largely determined by the functional groups present. In biological systems, the important properties of amino acids include the following: 1. **They can join to form proteins.** The average molecular weight of an amino acid is about 135kD. Proteins have molecular weights ranging from about 6,000 to several million. Thus, a large number of amino acids must be joined together to produce a protein. 2. Glycine, alanine, valine, serine, tryptophan, histidine and proline are sweet in taste; leucine is tasteless; while isoleucine and arginine are bitter 3. **All amino acids except glycine are asymmetric.** The carbon atoms to which the amino and carboxyl group is attached is called the alpha carbon. The mirror image forms produced with reference to the alpha carbon atom, are called D and L isomers. 4. 5. **Amino acids can act as either as an acid or base.** A zwitterion can act either as an acid when it donates an hydrogen atom/proton Or as a base when it accepts an hydrogen atom/proton 6. **Isoelectric point (pI).** At certain pH, amino acid is in its fully ionized form or as a zwitter ion. This pH is known as the isoelectric pH or point. **At a pH below the isoelectric point, some of the carboxylate groups will be protonated.** The pH required to cause this protonation of the carboxylate group depends on the dissociation constant Ka of the acid. Thus, the pKa of the carboxylic acid group is important**. At a pH above the isoelectric point, some of the ammonium groups will be deprotonated.** The pH required to cause this deprotonation of the ammonium group depends on the Ka of the ammonium group. **At its isoelectric point the, amino acid molecules will not move when placed in an electric field. The separation technique called electrophoresis relies on molecules with different isoelectric points moving at different speeds when kept at a fixed pH and placed in an electric field.The isoelectric point is calculated by averaging the pKa values for the carboxylic acid and the amine group.** 7. **Structural Formulae/classification of amino acids** **Amino acids can be classified using different criteria, these include** 1. **Polarity of R group** 2. **Chemical properties of R group** 3. **Nutritional needs** 4. **Metabolic fate of amino acids** **UNCOMMON AMINO ACIDS** **i. Derived amino acids found in proteins: After the synthesis of proteins, some of the amino acids are modified, e.g. hydroxy proline and hydroxy lysine are important components of collagen. Gamma carboxylation of glutamic acid residues of proteins is important for clotting process In ribosomal proteins and in histones, amino acids are extensively methylated and acetylated.** **ii. Derived amino acids not seen in proteins (Nonprotein amino acids): Some derived amino acids are seen free in cells, e.g. Ornithine , Citrulline, Homocysteine. These are produced during the metabolism of amino acids. Thyroxine is also as derived from tyrosine.** **iii. Non-alpha amino acids: Gamma amino butyric acid (GABA) is derived from glutamic acid. β - alanine, where amino group is in beta position, is a constituent of pantothenic acid (vitamin) and Co-enzyme A.** **PEPTIDES/PEPTIDE BOND FORMATION** **The interaction of amino acids at the body's pH results in the** **formation of a peptide bond. To form protein, the amino acids undergo condensation reaction between α-carboxyl group of one amino acid and α-amino group of another amino acid leading to the loss of water molecule. The chain of amino acids is also known as a polypeptide. A peptide chain consisting of two amino acid residue is known as dipeptide, three amino acids as tripeptide, four as tetrapeptides etc.**  aminacid **Basic principle of test for amino acids** 1. **Xanthoproteic Test:** **Aromatic groups of either the free amino acid or protein, undergo nitration on heating with concentrated nitric acid. The salts of these derivatives are orange in colour. It is a positive test for benzene rings which have amino or hydroxyl groups, such as that present in tryptophan and tyrosine** 2. **Cysteine Test:** ** positive test for cysteine** ** addition of cyanide salt to reduce disulfide bonds** ** addition of nitroprusside to from a colored complex with sulfhydryl groups** ** gives a pink to purple color intensity increases with concentration of --SH groups** ** if no nitroprusside is used a black precipitate will indicate positive test** 3. **Biuret Test: positive test for peptide bonds. Biuret Reagent is made up of sodium hydroxide and copper sulfate blue reagent turns violet in the presence of proteins pink color persists with short-chain polypeptides color intensity varies with concentration of peptides** 4. **Ninhydrin Test: This is a positive test for amino acids, not peptides. All amino acids give blue colour except proline which gives a yellow colour**  5. 6. 7\. Sulfur Test for Cysteine: When cysteine or cysteine-containing proteins are boiled with strong alkali, organic sulfur splits and forms sodium sulfide, which on addition of lead acetate produces lead sulphide as a black precipitate. Methionine does not answer this test because sulfur in methionine is in the thioether linkage which is difficult to break. Albumin and keratin will answer sulfur test positively; but casein will give a negative test. 8\. Nitroprusside Reaction for SH groups: Proteins with free sulfhydryl groups give a reddish color with sodium nitroprusside, in ammoniacal solution. Many proteins give a negative, reaction in the native state, but when denatured, reaction will be positive, showing the emergence of free SH groups. 9\. Pauly's Test for Histidine or Tyrosine: Diazo-benzene sulfonic acid reacts with imidazole group of Histidine to form a cherry-red colored diazotized product under alkaline conditions. The same reagent will give an orange red colored product with phenol group of Tyrosine. I. **What are proteins ?** II. **Classification of proteins** III. **Functions of proteins** ------------------- ---------------------------------------------- ----------------------------------------------------------------------------- **Role** **Examples** **Functions** Digestive enzyme Amylase, lipase, pepsin Break down nutrients in food into small pieces that can be readily absorbed Transport Hemoglobin Carry substances throughout the body in blood or lymph Structure Actin, tubulin, keratin Build different structures, like the cytoskeleton Hormone signaling Insulin, glucagon Coordinate the activity of different body systems Defense Antibodies Protect the body from foreign pathogens Contraction Myosin Carry out muscle contraction Storage Legume storage proteins, egg white (albumin) Provide food for the early development of the embryo or the seedling ------------------- ---------------------------------------------- ----------------------------------------------------------------------------- **PROTEIN STRUCTURE** **Protein function is directly related to its molecular structure, there are four structural levels proteins can assume. These are the primary, secondary, tertiary and quaternary.** A. **Primary structural level: The sequence of amino acids in each polypeptide or protein is unique to that protein, this is called the primary structure. If even one amino acid in the sequence is changed, that can potentially change the protein's ability to function.** **The figure below shows the primary structure of a tripeptide containing three alanine amino acids.**  **Amino acid residues in polypeptides are named by changing the suffix "-ine" to "-yl", e.g. Glycine to Glycyl. Thus peptide bonds formed by carboxyl group of glycine with amino group of Alanine, and then carboxyl group of Alanine with amino group of Valine** **and is called glycyl-alanyl-valine.** **A longer polypeptide primary structure**  **Each peptide chain will have an amino end and a carboxylic acid end, each amino acid is referred to as a residue. So, the ends are named, n-terminal residue and c-terminal residue or the n-terminus and c-terminus.** B. **Secondary structural level:This denotes the configurational relationship between residues, which are about 3--4 amino acids apart in the linear sequence. It is possible for one peptide bond to form a hydrogen bond to another peptide bond, the formation of these hydrogen bonds leads to the formation of secondary structure of a protein. The secondary structure is the result of many hydrogen bonds, not just one. The hydrogen bonds are intramolecular, between segments of the same molecule.** **This will cause the protein to have areas of its shape conforming to one of 3 shapes, alpha-helix, beta-pleated sheet or woven and collagen helix. The alpha-helix, beta-pleated sheet are the most common in proteins.** i. **The α-helix : It is a spiral structure, consisting of a tightly packed, coiled polypeptide backbone core, with the side chains of the component amino acids extending outward from the central axis to avoid interfering sterically with each other. A very diverse group of proteins contains α-helices. For example, the keratins are a family of closely related, fibrous proteins whose structure is nearly entirely α-helical. They are a major component of tissues such as hair and skin, In contrast to keratin, myoglobin, whose structure is also highly α-helical, is a globular, flexible molecule.** ii. **The β-sheet secondary structure is formed when all of the peptide bond components are involved in hydrogen bonding. The surfaces of β-sheets appear "pleated," and these structures are, therefore, often called "β-pleated sheets."** **A β-pleated sheet forms when two or more strands link by hydrogen bonds. The strands are different parts of the same primary structure.** - **Hydrogen bonds: It is a weak electrostatic attraction formed between hydrogen attached to an oxygen or nitrogen and the lone pairs found on an oxygen or nitrogen. Hydrogen atoms can be donated by -NH (imidazole, indole, peptide); -OH (serine, threonine) and -NH~2~ (arginine, lysine). Hydrogen accepting groups are COO--- (aspartic, glutamic) C = O (peptide); and S--S (disulfide).** - **Electrostatic bonds (ionic bonds): Positive charges are donated by epsilon amino group of lysine, guanidinium group of arginine and imidazolium group of histidine. Negative charges are provided by beta and gamma carboxyl groups of aspartic and glutamic acids.** - **Hydrophobic bonds are formed by interactions between nonpolar hydrophobic side chains by eliminating water molecules. This serves to hold lipophilic side chains together.** - **The van der Waals forces are very weak, but collectively contribute towards the stability of protein structure** **ii.**  **α-helix β-sheet** C. **Tertiary structure of proteins: The tertiary structure of proteins represents overall folding of the polypeptide chains i.e., further folding of the secondary structure. The interactions between the side chains rise to two major molecular shapes namely fibrous and globular. The main forces which stabilise the 2° and 3° structures of proteins are hydrogen bonds, disulphide linkages, van der Waals and electrostatic forces of attraction. Nonpolar side chains are hydrophobic and, although repelled by water, are attracted to each other. Polar side chains attract other polar side chains through either dipole-dipole forces or hydrogen bonds.** D. **Quaternary structure of proteins: Some of the proteins are composed of two or more polypeptide chains referred to as sub-units. The spatial arrangement of these subunits with respect to each other is known as quaternary structure.** **The quaternary structure found in some proteins results from interactions that stabilize both secondary and tertiary protein structure. This quaternary structure locks the complex of proteins into a specific geometry. An example is hemoglobin, which has four polypeptide chains.**  **Denaturation of proteins** **Protein denaturation results in the unfolding and disorganization of the protein\'s secondary and tertiary structures, which are not accompanied by hydrolysis of peptide bonds. When a protein is destroyed it is said to be denatured. Certain conditions will cause the protein to unfold, leaving only the primary structure. They are** ** Heat -- breaks hydrogen bonds by causing the atoms to vibrate too radically** ** UV light -- breaks hydrogen bonds by exciting bonding electro3ns** ** Organic solvents -- breaks hydrogen bonds** ** Strong acids and bases -- breaks hydrogen bonds and can hydrolyze the peptide bonds, breaking the primary structure** ** Detergents -- disrupt hydrophobic interactions** ** Heavy metal ions -- forms bonds to sulfur groups and can cause proteins to precipitate out of solution.** **Denaturation may, under ideal conditions, be reversible, in which case the protein refolds into its original native structure when the denaturing agent is removed. However, most proteins, once denatured, remain permanently disordered. Denatured proteins are often insoluble and, therefore, precipitate from solution** **STRUCTURE OF MYOGLOBIN AND HEMOGLOBIN** **Myoglobin and hemoglobin are globular proteins that serve to bind and deliver oxygen. Myoglobin is found in vertebrate muscle cells. Muscle cells, when put into action, can quickly require a large amount of oxygen for respiration because of their incredible demand for energy. Therefore, muscle cells use myoglobin to accelerate oxygen diffusion and act as localized oxygen reserves for times of intense respiration.** **In vertebrates, hemoglobin is found in the cytosol of red blood cells in the bloodstream.** **Myoglobin is a monomeric heme protein, during periods of oxygen deprivation oxymyoglobin releases its bound oxygen which is then used for metabolic purposes. Its secondary structure is unusual in that it contains a very high** **proportion (75%) of α-helical secondary structure. A myoglobin polypeptide is comprised of 8 separate right handed alpha-helices, designated A through H, that are connected by short non helical regions. The tertiary structure of myoglobin is that of a typical water soluble globular protein.** **Hemoglobin and myoglobin are only slightly related in primary sequence. The secondary structures of myoglobin and the subunits of hemoglobin are virtually identical.** **Both proteins are largely alpha‐helical, and the helices fit together in a similar way.** **Hemoglobin is an \[α(2):β(2)\] tetrameric hemeprotein found in erythrocytes where it is responsible for binding oxygen in the lung and transporting the bound oxygen throughout the body where it is used in aerobic metabolic pathways. Each subunit of a hemoglobin tetramer has a heme prosthetic group identical to that described for myoglobin. Although the secondary and tertiary structure of various hemoglobin subunits are similar, reflecting extensive homology in amino acid composition, the variations in amino acid composition that do exist impart marked differences in hemoglobin\'s oxygen** **carrying properties. In addition, the quaternary structure of hemoglobin leads to** **physiologically important allosteric interactions between the subunits, a property lacking in monomeric myoglobin which is otherwise very similar to the α-subunit of hemoglobin.** **Differences between myoglobin and hemoglobin** 1. Hemoglobin transports oxygen in blood while myoglobin transports or stores oxygen in muscles. 2. Myoglobin consists of a single polypeptide chain and hemoglobin consists of several polypeptide chains. 3. Unlike the myoglobin, concentration of hemoglobin in red blood cell is very high. 4. At the beginning, myoglobin binds oxygen molecules very easily and lately become saturated. This binding process is very rapid in myoglobin than in hemoglobin. Hemoglobin initially binds oxygen with difficulty. 5. Myoglobin occurs as a monomeric protein while hemoglobin occurs as a tetrameric protein. 6. Two types of polypeptide chains (two α-chains and two β- chains) are present in hemoglobin. 7. Myoglobin can bind one oxygen molecule so called monomer, while hemoglobin can bind four oxygen molecules, so called tetramer. 8. Myoglobin binds oxygen more tightly than does hemoglobin. 9. Hemoglobin can bind and offload both oxygen and carbon dioxide, unlike the myoglobin.