CHEMISTRY OF PROTEINS CHAPTER 6 Major Concepts ‘A. Toknow what are protoins and their biomedical importance. 1B Toloan wnat are amine acs. ter classification ana popertes. C._Tolearn the classiicaton and properties of proteins Lear the structure of protein, Specitic Objective A. Daline pate, AND AMINO ACIDS 2_Descibe the biomedical importance of protein and learn compostion of proteins, 8, Basic monomeric unit of protin is amino acd. Classify amino acids {eam the nonstandard amino acts. eam the occurence of amin acids. Lear he general funcions of amino acs. [Lear the physical and chemical properties of amino acids Classy proteins, + Based on size and shape, 1 2 3 4 5 6 What are amino acids? Learn te basic uct of amino acid Unt essential amino acide, semiessental aminoacids and non-essential amino acids and why they ae called eo. + Based on funcions, + Based on solubility, structure and physical properies—Most commonly ‘employed Gassiicaton According to ths proteins are classifled as simple, corjugated and dived proteins. ‘Learn bw physical and chemical properties of roti. Learn preiptaton reaction of protins and ts applicator ‘Study the primary structure of protein. Lean varios colour ractions of protein de o specie aminoacid Learn te peptide linkage in apron mecule and earn few biologically important peptides. ‘Study the secondary sirvcture of pati, knkagos and types such as «hol, pleted sheet suture, Tiple helix, and Random col Lear the tertiary suture, bonds Imeved intertary structure formation. Lear he quaternary structure, bonds that make it and examples. \Winat is denaturation of pectin? Learn various factors that cause denaturation, ts application and the changes a protein molecule ‘undergoes ater denaturation + Study the crtria of purty of prot, In 1839 Dutch chemist GJ Mulder while investigating substances such as those found in milk, egg, found that they could be coagulated on heating and were nitroge- nous compounds. Swedish scientist JJ Berzelius suggested to Mulder that these substances should be lled proteins. The term is derived from Greek word Proteios me holding first place” or “pre-eminent” be htthem tobe most important of biological substances. And now we know that proteins are fundamental structural components of the body. They are nitrogenous “macromolecules” composed of many amino acids. Proteins are the main structural components of the ‘ytoskeleton. They are the sole source to replace Nitrogen ‘ofthe body Biochemical catalysts known as enzymes are proteins. Proteins known as immunoglobulins serve as the it ine ‘of defence against bacterial and viral infections. Several hormones are protein in nature. ‘Structural proteins furnish mechanical supportand some ‘of them lke actin and myosin are contractile proteins and hlp in the movement of muscle fibre, microvilli, ot. ‘+ Some proteins present in cell membrane, cytoplasm and ‘nucleus ofthe cell act as receptors.+ The transport proteins carry out the function of wans- porting specie substances eter across the membrane rine body fis. + Storage proteins ind wih specie substances and store thom, eon is stored as ferrin. + Fow proteins are constvers ol respiratary pigments and ‘o2curin electron tanspot chain o respiratory chain 9 ytochromes, hemoglobin, myosabin. + Under crtain conatons pratmns canbe eatabolised to supply energy. + Proteins by means of exerting osmotic pressure help in martenance of elect and water balance n body Poesia) In addition to C, H, and O which are present in carbo- hydrates and lipids, proteins also contain N. The nitrogen content is around 16 per cent of the molecular weight of. proteins, Small amouints of Sand P are also present. Few other elements such as 1, Cu, Mn, Zn molecules are with a high molecular weight ranging from 5000 to 25,00,000. Protein can be broken down into smaller units by hy is. These small units the monomers of protei ino acids, Proteinsare made up from,20such standard amino acids in differentsequences and numbers. oan indefinite numberof proteins can be formed and do occur in nature. Thus proteins are the unbranched polymers of L- a-amino acids. The L- amino acid has a general formula as shown, below: Ris called a side chain and can be a hydrogen, aliphatic, aromatic or heterocyclic group. Each amino ‘has an amino group -NH,, a carboxylic acid growp ‘OOH and a hydrogen atom each attached to carbon located next to the - COOH group. Thus the side chain varies from one amino acid to the other. ‘AMINO ACIDS Creer) | STRUCTURE OF AMINO ACIDS ‘Amino acids can be classified into 3 groups depending fn their reaction in solution, A. Neutral B. Acidicand Chemistry of Proteins and Amino Acids 77 A. Neutral amino acids: This is the largest group of amino acids and can be further subdivided into aliphatic, aromatic, heterocyclic and S-containing amino acids. ic Amino Acids: Gly) oF a-amino acetic acid. e008) 4] (optcaly inactive) 2. Alanine (Ala) or a-amino propionic acid. opm 3. Valine (Val) oF a-amino-isovaleric acid. So isocaproic acid. 5. Isoleucine (Ile) oF a-amino-B-methyl valeric acid. Bs ws Sa Me pe i Alloftheabove are simple monoamine monocarboxy- lic acids. The next from the neutral group of amino acids are iydroxy amino acids. Since they contain-OH group, in thei side chains. acid, 6. Serine (Ser) oF a-amino-f-hydroxy propionic78 Chemistry of Biomolecules 7. Threonine (Thr) or a-amino-B-hydroxybutyric acid. (b) Aromatic Amino Acids [Second subgroup of neutral amino acids consists of aromatic amino acids. 8. Phenylalanine (Phe) or id. a | ar i — (G0 4 9. Tyrosine (Tyr) or parahydroxy phenylalanine or ccamino-frparahydroxy phenylpropionic acid. ny wore (©) Heterocyclic Amino Acids: Third group belongs to heterocyclic amino acids. 10. Tryptophan (Trp) or a-amino-B-3-indole propion facid. This amino acid is often considered as aromatic amino acid since it has aromatic ring in its structure. sino-B-phenyl propionic nA 11. Histidine (His) or a-amino-B-imidazole propionic acid. ° “Ty Histidine is basic in solution on account of the imidazole ring and often considered as Basic Amino acid. (@) Imino Acids 12, Proline (Pro) or Pyrrolidone-2-carboryl (Jeon wh ¥ acid. 13, Hydroxyproline (Hyp) or 4 hydroxy pyrrolidone-2 5 Jes Proline and Hydroxyproline do not have a free-NH, group but only a basie pyrrolidone ring in which the Nitrogen of the Imino group is in a ring but can still function in the formation of peptides. These amino acids are therefore called as imino acids. (€)'S' containing amino acids: The fourth subgroup of neutral amino acids contains two sulphur contain amino acids. 14, Cysteine (Cys) or a-amino-B-mercaptopropionic acid. (=) Wy b,—t oon ecae ‘Two molecules of cysteine make cystine (eys-cys) or dithio B, Ba aminopropionic acid. The S-S linkage is called as disulphide bridge. 7 ae SB 15, Methionine (Met) or a-amino ymethylthio-n- butyric acid. B. Acidic amino acids: These amino acids have two ~COOH groups and one~NH, group. They are therefore ‘monoamtinodicarboxylic acids. 16, Aspartic acid (Asp) or a-amino succinic acid,Asparagine (Asn) or y-amide of a-aminosuecinic acid. 17, Glutamic Acid (Glu) or a-aminoglutarie acid. CHy—cHy— 4 Glutamine (Gln)-Amide of Glutamic Acid or &-amide of @-aminoglutaric acid. oy yo fama ‘ C. Basic amino acids: This class of amino acids consists, ‘of those amino acids which have one ~COOH group and. two-NHT, groups. Thus they are diamino monocarboxy- licacids, Arginine, lysine and hydroxylysine are included in this group. 18. Arginine Arg) or a-amino-& guanidino-n-valericacid. 19, Lysine (Lys) or a- e-diamino caproic acid. 2 6 7 8 I fice Oo " 20. Hydroxylysine (Hyl) or a--diamino-6-hydroxy-1 valeric acid, Gertie Oh of (8H) [onl " As already mentioned histidine is also classified as basic amino acid. Chemistry of Proteins and Amino Acids 79 Non-standard Amino Acids A. The compounds similar to basic structure of amino acids but do not occur in proteins. Examples of some of those are: Baalanine: They found in coenzyme A. Taurine: They found in bile acids Ornithine and citrulline: They are intermediates in urea cycle ‘Thyroxine (T,) and Tri-iodo Thyronine (T,): Thyroid. hormones synthesised from tyrosine. aminobutyric acid (GABA): A neurotransmitter produced from glutamic acid, Beamino isobutyric ac pyri 5-aminolaevulini mediate in haem synthesis. S-adenosyl methionine (SAM): These are methyl I: These are end product of These are inter- donor formed from L-methionine 3,4-dihydroxy phenyl alanine (DOPA): A precursor ‘of mela nine pigment. B. D-amino acids: These are non-standard amino acids— ‘Amino acids normally isolated from animal and plants are L-amino acids. But certain D-amino acids are found in bacteria and antibiotics and in brain tissues of animals. Deglutamic acid and D-Alanine are constituents of bacterial cell walls. D-amino acids are found in certain antibiotics, eg, gramicidin-S, Actinomycin-D. Animal tissues contain L-amino acids which are ‘deaminated by Lamino acid oxidase. But there is also present D-amino acid oxidase the function of which ‘was not known. Now D-amino acids like D-aspartate and D-serine have been found in brain tissue. This ‘explains the existence of D-amino acid oxidase. ‘Apart from being the monomeric constituents of proteins and ‘peptides, amino acids serve variety of functions. {(@) Some amino acids are converted to carbohydrates and are called as glucogenic amino acids. (8) Spectic amino acids give rise o specialised products, 6g ‘+ "Tyrosine forms hormones such as thyroid hermones. (Ty, T.). epinephrine and norepinephrine and a pigient called melanin, “Tryptophan can synthesise a vitamin called niacin, Glycine, arginine and methionine synthesise creatine. Glycine and cysteine help in synthesis of Bile sats. ‘Glutamate, cysteine and gycine synthesise glutathione. Histidine changes to histamine on decarboxyiation, ‘Serotonin is formed from tryptophan. Glycine is used for the synthesis of haem. Pytimidines and purines use several amino acids for ‘their synthesis such as aspartate and glutamine for pyrimicines and glycine, aspartic acid, Glutamine and ‘serine for purine synthesis.80 Chemistry of Biomolecules {€) Some amino acids such as glycine and cysteine are used ‘as detoxicants of specc substances. (@) Methionine acts as “activ” methionine (S-adenosy/- ‘methionine) and transfers methy! group to various ‘substances by transmethyation. (©) Cystine and methionine are sources of sulphur. Essential Amino Acids ‘Nutritionally, amino acids ae of two types (a) Essential Jand (b) Non-essential.(c) There is also a thd group of [semicessential amino acids. {@) Essential amino acids: These are the ones which ar not synthesised by the body and must be taken in diet. ‘They include valine, leucine, isoleucine, phenylalanine, threonine, tryptophan, methionine and lysine. For remembering the following formula is used—MATT VIL PuLy. (b) Non-essential amino acids: They can be synthesised by the body and may not be the requisite components of the diet. | amino acids: These are growth promoting factors since they are not synthesised in sufficient quantity during growth. They include arginine and histidine. They become essential in growing children, pregnancy and lactating women. Occurrence of amino acids: All the standard amino acids ‘mentioned above occur in almost all proteins. Cereals are rich in acidic amino acids Asp and Gh while collagen is rich in basic amino acids and alsa proline and hydraxy- proline. New Amino Acids In addition to 20 L-amino acids that take part in protein synthesis, recently two more new amino acids described, They are: A. Selenocysteine - 21st amino acids B. Pyrrolysine - 22nd amino acid A. Selenocysteine Selenocysteine is recently introduced as 21st amino acid. Selenocysteine occurs at the “active site” of several enzymes. ‘+ Glutathione peroxidase which scavenges peroxides, + De-iodinase that converts thyroxine to trie iodothyronine Glycine reductase Selenoprotein P, a glycoprotein containing 10 selenocysteine residues, found in mammalian blood. It has an antioxidant function and its concentration falls in selenium deficiency Selenocysteine arises co-translationally during its incorporation into peptides. The UGA anticodan of the unusual tRNA designated tRNA", normally signals sop. The ability of the protein synthesising apparatus to identify a selenocysteine specific UGA. codon involves the selenocysteine insertion element, a stem-loop structure in the untranslated region of the m-RNA. ‘Sclenocysteine-RNA"is first charged with serineby the Ligase that charges tRNA". Subsequent replacement of the serine oxygen by selenium involves seleno- ‘phosphate formed by Selenophosphate synthetase. i se— ots —}—c00 NHS? (Selenonysteine) Se OATP AMP +P 80 —F-o" o Reaction catalysed by slenophosphate synthetase Ina simpler way, the reaction that occurs Selenophosphate ‘sminease Se+a1p ————+ so_P+aMe +P) Serna + Se P+ Seoys +P (Gelenooysoine) B. Pyrrolysine—the 22nd Amino Acid Recently ithas been claimed as 22nd amino acid by some scientists. The STOP codon UAG can code for pyrrolysine. Gta A. Isomerism: Two types of isomerism are shown by amino acids basically due to the presence of asymmetric won atom. Glycine has no asymmetric carbon atom in its structure hence is optically inactive. (a) Stereoisomerism: Allamino acids except glycine exist inD and L isomers. As described in the chapter on carbo hydrates itis an absolute configuration. In D-amino acids = NH, group is on the right hand while in L-amino acids itis oriented to the left. Itis the same orientation of -OH ‘group of the central carbon of glyceraldehyde (Fig. 6.1). LAming cia D-Amino acid Fig. 6.1: and Dorms of amino acdNatural proteins of animals and plants generally contain L-amino acids. D-amino acids occur in bacteria. (b) Optical Isomerisim: All amino acids except glycine have asymmetric carbon atom. Few amino acids like ‘soleucine and threonine have an additional asymmetric carbon in their structures. Consequently all but glycine exhibit ‘optical’ activities and rotate the plane of plane polarised light and exist as dextrorotatory (d) or Inevorotatory (D) isomers. Optical activity depends on the pHand side chain, B. Amphoteric Nature and Isoelectric pH: The -NH, and -COOH groups of amino acids are ionizable groups. Further, charged polar side chains of few amino acids also ionise. Depending on the pH! of the solution these {groups act as proton donors (acids) or proton acceptors (bases). This property is called as amphoteric and therefore amino acids are called as ampholytes. At a specific pH the amit no acid carries both the charges i equal number and exists as dipolar ion or *Zzvitterion”” 'At this point the net charge an it is zer0, i.e. positive charges and negative charges on the protein /amino acid molecule equalizes. The pH at which it occurs without any charge on it is called pl o isoelectric pH. On the acidic side of its pl amino acids exist as a cation by accepting a proton and on alkaline as anion by donating, a proton. . Physical Properties: They are colourless, crystalline substances, more soluble in water than in polar solvents. ‘Tyrosine is soluble in hot water. They have high melting, point usually more than 200°C. They have ahigh dielectric constant, They possess a large dipole moment, D. Chemical Properties 1. Due to Carboxylic (—COOH) Group 1. Formation of esters: They can form esters with alcohols. The COOH group can be esterified with alcohol Treatment with Na,CO, solution in cold releases the free ester from ester hydrochloride. 2. Reduction to amino alcohol: This is achieved in pre- ‘ence of lithium aluminium hydride. 3. Formation of amines by decarboxylation: Action of specific amino acid decarboxylases, dey distillation o heating with Ba(OH), or with diphenylamine evolves CO, from the COOH group and changes the amino acid into its amine (Fig, 6.2). In vivo, the amino acids can be decarboxylated by the enzyme decarboxylase and forms the corresponding 1 eR cH, — Ny ming nid Fig. 62: Formation of amine (decarboryiaion) mine Chemistry of Proteins and Amino Acids 81 4. Formation of amides: Anhydrous NH, may replace alcohol from its combination with an amino acid in an ‘amino acid ester so that an amide of amino acid and a Inolecue of ee cools produced ig 63) We a a beim, ——s e—orecnon 72 acon bocca k \, E = = jamrasee ano scdenise FY rs Fig. 63: Formation of amide 1 Properties Due to Amino (-NH,) Group 1. Salt formation with acids: The basic amino group reacts with mineral acids such as HCL to form salts like hydrochlorides (Fig. 6.4). 008 00H HyC—NMy + HC! > Hjc—Nic1 Giycine Glycine hydrochloride Fig. 6.4: Salt foxmatin of amino acd 2. Formation of acyl derivatives: Amino group reacts, ‘with acyl anhydride or acyl halides such as benzoyl Chloride and give acyl amino acids like benzoyl glycine (hippuric acid). Incidentally, this is one of the ‘mechanisms of detoxication in which glycine is used and this also forms the basis of one of the liver function tests. 3. Oxidation: Potassium permanganate or H,O, oxidises ‘the NH, group and converts the amino acid into imino acid which reacts with water to form NH, and acketo- acid. Like other primary amines, the ‘amino acids except proline and hydroxyproline react, ‘with FINO, (nitrous acid) libering N, from NH, group. This forms the basis of Van Slyke’s method for determining NH, group (Nitrogen) 5. Reaction with CO. The amino acid anion present in an alkaline solution may react with CO; through NH ‘gr0up to form a carbaminoacid anion, 6. Reaction with formaldehyde: Formaldehyde reacts ‘with the-NH, group to form a methylene compound. Application: Because of the presence of free basic ‘amino group in the amino cannot be estimated directly by titration with a ‘standard alkali. On addition of neutral formaldehyde it combines with the amino group to form either ‘methylene amino acid or dimethylol amino acid. Both these products are strong acids and may be estimatedSECTION TWO on | | H oydroxy acid Fig. 65: Reaction wih HNO, ‘COOH Fig. 6.6: Reaction of gycne with frmalderyde-Basis of Sorensen’ ermel tration by titration with a standard alkali. This is known as “Sorensen’s” formol titration method (Fis. 6.6) 7, Specific colour reactions: Reactions with Ninhydrin, Millon’s test, Sakaguchi test, Hopkins-Cole test are discussed under properties of proteins Identification of N-terminal Residue (a)N-terminal residue can be identified by using a reagent that bonds covalently with its o-NH, group. Because the bond is stable to hot acid hydrolysis, the derivative of the N-terminal residue can be identified by chromatographic procedures after the protein has been hydrolysed. ‘Two reagents are commonly used 1. Sanger’s reagent: The reagent contains 1-fluoro-2, 4-dinitrobenzene (FDNB). Itreacts with free -NH, group in an alkaline medium. ond Sl pe i Nr} ata onl Sepa i The reaction can also take place with the N-terminal, “NIL, group of the polypeptide chain. The compound so formed can be isolated after protein hydrolysis and identified. Sanger was first to sequence a polypeptide. He determined the complete primary structure of the hormone insulin, 2. Reaction with Dansyl Chloride: The N-terminal -NH, group can also combine with Dansyl chloride i rose fame secen ‘ (1-dimethyl aminonaphthalene-5-sulphonyl chloride) to forma fluorescent dansyl derivative which can be isolated. and ident (b) Ediman reaction: A similar reaction with NH} group ‘can occur with the reagent phenyl isothiocyanate and thus | enables the identification of the N-terminal amino acid (Refer box in right side). ‘Sequenator Edman and G. Begg have perfected an automated amino acid sequenator for carrying out sequential degradation of peptides by the phenylisothiocyanate procedure (Edman’s reaction). Automated aminoacid sequencers now widely used, which permit very rapid determination of the amino acid sequences of polypeptides upto 100 amino aci approximately ‘Amino acids are determined sequentially from. ‘N-terminal end. The phenyl thiohydantoin amino acid liberated is identified by high performance liquid chromatography (HPLC).Chemistry of Proteins and Amino Acids 83 IN, Properties of Amino acids Due to Both NH, and COOH Groups In addition to the property of reacting with both cation and anion, the amino acids form chelated, co-ordination complexes with certain heavy metals and other ions. ‘These include Cur*, Co", Mn** and Ca**. An example of chelated complex of Ca and glycine is given in Figure 6.7. Eiieaa eu CChelates are non-ionic and therelore amino acids may be used to remove calcium from bones and teeth. tis possible that the amino acids resulting from the breakdown of enamel ‘and dentine coud inthis way form soluble calcium complexes thereby causing a loss of calcium and the development of caries. PROTEINS Geter TL. On the basis of functional properties IIL. On the basis of solubility and physical properties. 1. On the basis of shape and size ++ Fibrous proteins: When the axial ratio of length: width of a protein molecule is more than 10, itis called a fibrous protein. Examples: arkeratin from hair, collagen. + Globular protein: When the axial ratio of length: width of a protein molecule is less than 10, it is called as globular protein. Examples: Myoglobin, haemoglobin, ribonuclease etc. IL,On the basis of functional properties: The second way of classifying proteins makes use of their functional Properties, such as: * Defence proteins: Immunoglobulins involved in defence mechanisms. + Contractile proteins: Proteins of skeletal muscle involved in muscle contraction and relaxation + Respiratory proteins: Involved in the function of respiration, like haemoglobin, myoglobin, cyto- chromes, + Structural proteins: Proteins of skin, cartilage, nal. ‘+ Enzymes: Proteins acting as enzymes. Hormones: Proteins acting as hormones. IIL On the basis of solubility and physical propert However, both the above classification schemes have many overlapping features. Therefore a third most acceptable scheme of classification of proteins isadopted, ‘According to this scheme proteins are classified om the basis of their solubility and physical properties and are divided in three different classes. A. Simple proteins: These are proteins which on complete hydrolysis yield only amino acids. B. Conjugated proteins: These are proteins which in addition to amino acids contain a non-protein group called prosthetic group in their structure. C. Derived proteins: These are the proteins formed from native protein by the action of heat, physical forces or chemical factors. A. Simple Proteins ‘These ae further subclassified based on their solubilities and heat coagulabilities. These properties depend on the size and shape of the protein molecule. Major subclasses of simple proteins are as follows: 1. Protamines ‘These are small molecules and are soluble in water, dilute acids and alkalies and dilute ammonia and non- coagulable by heat. They do not contain cysteine, tryptophan and ‘but are rich in arginine. Theit isoelectric pH is around 7.4 and they exist as basic proteins in the body. They combine with nucleic acids to form nucleoproteins. Examples: Salmine, sardinine and cyprinine of fish (sperms) and testes. 2. Histones These are basic proteins, rich in arginine and histidine, with alkaline isoelectric pH. They are soluble in water, diluteacids and salt solutions but insoluble in ammonia, They do not readily coagulate on heating. They form conjugated proteins with nucleic acids (DNA) and porphyrins. They act as repressors of template activity of DNA in the synthesis of RNA. The protein part of hemoglobin, globin is an atypical histone having predominance of histidine and lysine instead of arginine. Examples: Nucleohistones, chromosomal nucleoproteins and globin of haemoglobin. For details of Histone case refer to chaper on chemistry of Nucleic acids. 3. Albumins These are proteins which are soluble in water and in dilute salt solutions. They are coagulable by heat and are