> Amino acids :— Intsoduction:- Amino acids epvesent one of the most impostont classes of naturally occusing compounds, because they fowmed fundamental stsuctural units of proteins. All naturally oceusing amino acids have an amino group located at «- position cirth respect +o the Coxboxglic gqsour- aH R- CH— CooH \ NH2 Psolme and hydwoxyproline are exceptions as in these molecules the amino group fowms a part of pywrolidine sing. HO . . Mi = a ‘oline <-OH Proline NN: G 3 Hy were i N: 4 ° T ' ° H In addition +o carlooxylic ond amino qeoups, An «- amino acid Some amino acids contain benzene or heterocylic xing system, phenolic of alcoholic group ov even Sulphur. Steucture ond Steveochernistyy: - — The steuctrse of a typical amino acids contains a Chirsality centve at the «-carbo.—The thvee dimensional and plane projection fosmulas of a typical wK-aMming acids ase shown below: - Wa re Reo —COOH ox H,N- cf H Ha R - AN amino acids ave Optically active except Glycine. because Gigeme does not centre. contain Chival oo Hn—C—H i Glycine — All the naturally occusing o- amino acids belong te L- series. because they have some relative configuration as L- Giyceval dehyde coon CHO eo ial = CHAOH L- Amino acid i= Glyceval dehyde — Most of amimo acids have S- configuration.—> Amino acids:- — Acid Base behaviour: Dipolas nature:+ — When Wwe added on omine in cashoxylic acid , it gives ammonium salt of Casboxylic acid . This takes Place by the teansfexs of a proton from the Comborylic qsoup te the amino qvoup. — In case of amino acids, both these qroups ore part of the same wolecule. In this situatom the proten transfers occurs intemalty to give on intemal salt called “dipolarion” ox "zwiiterion". R-CH— COOH ——— Rach coo \ NH, @nu, An &- amino acid zwitter ion — Evidence in favour of the dipolas Ionic ctructare?- — Because of the dipolox jonic Structure, amino acids Show the following typical properties :- @) They ase highly soluble in water ond insoluble in common organic solvents- Gy They melt at +ermp- above 473K and that too with decomposition. Gi) Theix aqueous Solutions behave like the solution of substace having dipole moment- Gv) The spectral studies do not show the presence of free -NHZ and -COoH groups but indicate the peesence of —Rs and -coo- groups. w) Because of dipolay natuse they ove amphoteric , i-e- » they can accept a proton Fesm stronger acid as well as donate a proton to a stronger base.- In aqueous solutions, the amino acids exist each other. Wates acts both as an acid and as a base. = R- CH— COOH —: @NH3 Ib R- CH- Coo! @®Nnv, Ta -ut [+H R-CH- COOH | NH I . —Ht jamie =—— Ht R—cH- coo \ NHa Te — The position of equilibrium depends Upon the pH of the solution. At (ower the Conjugate base (1b) predominates Lohile at highew pH Cie. in alkaline base (1c) preodominates- im Various species Which are in equilibhium with ph (é-e- ln acidic solution) solution) the conjugateAmine acids: 4 > Nomenclature .7 — The common names of all amino adds are given on the basis of theix source of theix special properties. or on the basis eg. Glycine +4 sweet taste 4 C&veck, Giykys, sweet) Leucine > It was isolated for the fixst time Som muscle fibers — (Greek, leucos vhite) Tysosine + obtained from caseine > (treek, tyvos, cheese) Cystine > Obtained from uBinary calculi ( Geek, kystis, bladder) ~The names of the awino acids are generally written in their abbreviated fowns. eg. Gigcine > aly Alanine 2 ala & ~ Classification: > 3 On the basis of position of amino group Gith wespect to Coxborylic group. — Amino acids ave classified as &- |B-,Y¥- amino acid etc. Fox example- & BK Yok, S NH, CH2- COOH NH,- CHy CH> COOH NES Na ig Sig COOH o- aminoacetic acid B-awinopropancic add Y- aminobutyric acid > On the basis of relative number of amino and Carborylic qrowps jw theiy molecule. G1 Neutral amino acids: Contain equal numbex of amino and carboxylic groups: oq: Glycine alanine , etc.i) Acidic amino acids:+ Contain ‘more Casboxylic groups as compared +o amino groups — Cartorylic geoup nay be present as free Carboxylic group ox Tn the form of amido group. €g- aspartic acid, glutamic acid , Osporagine , etc. Wi) Basic amino acidsis Contain more amino groups as compared to corkoyic qroups- eq lysine , axginine , etc. > On the basis of wequisemment +o the human body :- © Non - essertial amino acids :> — The arrimo acids, Which can be synthesised inthe body are known as Non- essential amino acids Gi) Essential amino acids :> - The amino acids which con not be synthesised in the kedy ave Known as essential amin acids. These amino acids youst be obtained through diet. Table :- Some common amino acids : | —cH —1__ . Valine* | —CH(CH,), @-a, COOH |. Leucine* —CH,CH(CH,), NHL ._Isoleucine* — CH(CH,)CH,CH, —* Essential amino acids 14. Asparagine 16. Proline 17. Hydroxyproline~ Tsoelectric point _of aynino acids: - The pH at which a pasticular amino acid exists as a dipolax ion is called the isoclectic point (pt). — The isoelectsic point of amino acids have no additional acidic ov basic groups ond is exactly half of the sum of pKa value of —cooH qsouP ond pka value of Bu, group. ® Isoelectsic point (pI) of a neutval awino acid = > Example :- Isoeleetsic point of Glycine ¢ NH cH-Coon). — In aqueous solution, glycine exists in the Following species Which are in equilibrium with each other & ae @ CH. coo- — CH,-— Coo NH,— CH— COOK j= Heo = =—=— NH,— — ih “eo a snp Ma” Che Ib To Ie Conjugate acid conjugate base — Now when the conjugate acid of glycine is titrated against alkali ond a qraph is Plotted With numbes of equivalents of base against pH of the solution these appear two equivalence points. fig: Titration curve of Glycine Equivalents of base added>—The tisst equivalence point indicates the chonge of Ib into Ta which coxresponds to pKa of d- Cavboxylic group (2:34) and is Several Units lower than that of on ordinary Carboxylic acids, ® - @ = i = NH3— CH= COOH ——» NH,— CH,— COO~ 5 NH, - CH{- COO Ib To Ie —Tthe second equivalence pomt represents the change Leom dipolar fowm Ia +o basic form Ic cowresponding to the pka of Q a&- bug gqroup (9-60) and is one umt lower than that of aliphatic ammorium fons. - After the addition of one equivalent of “NoOH thete Is a steep xise in pH of solutim and the curve tokes the shope as shown in fig. This point Cowresponds to pH 9°37 at which the ada exists as dipoles ion. This point is cated jJsoelecteic pot of Gycine at ohich the sum of chayges of all the molecules of amino acid is zevo. Isoelectsic point of Gigeme = wee + 9:97 - Iscelectsic pomt can also be defined as the pH at which the concentration of Ib is exactly equal to that of Ic. —In genevol, pt of amino acids Iie between 4 ard 8 When they exist as dipolar im. — Isoelectsic point is characteristic Property of amino acids. tf aon electic cument is passed theough the aqueous solution of amino acid at pH lower than pT, the armino atid will miguate +owasds cathode. Similarly at higher PH it will migrate towards Onode- But at pH equal to pt there will no migsotion because of the existarce of amino acid as dipolar ion.- Isoelectsic point May also be defined as the pH at Which amino acid does not Migrate tohen its aqueous solution placed undex the influence of an electsic field. — The solubility of amino acid is yinimum at it isoelectvic poimt, and this property has been quite useful in the separation of amino acids fem Yixtures .~> Electrophoresis :> — Electsophovesis is a technique of separation and purification of compounds on the basis of diffesential movement of chaxged particle in an electvical field. — This technique is used for separation of different amino acids obtained on hydvolysis of 0 protein > Methodology: — A paper Stvip os @ Suitable plastic ox cellulose acetate plate ts used in this technique. - In poper electrophowesis, the rnixture of amino acids is place in the fowm of spot at the center of the papes stwvip- — The stip is then socked with an aqueous buffer of a particulae pH. The pH of the buffer depends upon the Jsoelectric points of the amino acids to be Separated. _ The two ends of the filtex popes ave dipped ‘ihe the buffer solution in Which the electyndes axe dipped- — when a electsic field ts applied, diffesent amino acids migrate towards cathode ox anode ot different rates depending Upon theix isoelectric Points and charge density. Ac a sesult , amino acids can be sepasated . The following chamges occur : G) The amino acids, whose isoelectsic point is the below pH of the buffer, Stast Moving towards Onode because they exist mainly in the anionic form - Gi) The amino ocids whose isoelectsic point is the above pH of the butler, ctast Moving dowords cathode because they exist moinly in the cationic foxm. diy The amino acids, whose [Soelectic point Corvesponds to the pH of the buffer, do not migrate from the ovigin because they have no net charge.— After the sepasation is over, the papew stvip is dried and Sprayed With a developer Such as Ninhydrin When different amino acids become visible- > Example:- Separation of mixture of Gilycine Cpl= 5-97), Aspartic acid CpI=2-98) and lysine Cpt= 9-7). - The pH of buffer Solution is 5-97 ~ At this pH, aspartic acid will move towards andoe, lysine will move towards cathode while glycine will not Yoigvate at all and hence remains at the origin point. Dy Filter paper soaked in buffer Solution. T Spot of the mixture Anode of amino acids @) Belose applying ‘voltage Arnino acid original Amion acid fA L - £ Sepogatady ) After applying Voltage Cathode figi- separation of amino acid wircture by papery electrophoresis.2 Amino acids:- > Methods of preparation:? 1. Feom proteins:> — Proteins fom vVavious sousces ave extracted with water, saline, acid, alkali and ethanol: — These extvacts so obtained ave subjected to dialysis to free from ionic impusities- — They they ave evaporated to dasyness. — The wesidue ts hydvolysed by Following method s- (@) Acidic medium:- By sefluxing with 6N Hci fox several hours. We) Alkoline medium:- By wefluxing With SN Ba(on),. «© Proteolytic enzymes:- Since , in acidic o€ basié mediums. some of the amino acids ave destroyed - So, the hydvely sis of these proteins is better accomplished by the use of certo proteolytic enzymes. This process , though quite slow, but does not involve destruct of ong of the constituent amino acids. — Te mixtute of amino acids obtained after hydwolysis is estevified and the mixture of estess is Separated by Fractional distillation. ~ Another Method its Dakins procedure. In this method, butanol- Water wixture is used for Selective extraction of amino acids- The *esidue is then separated by using special Yeagents such as Phosphotungstic acid. - The separation of ammo acids may also achieved by electvophoxesis , jon exchange , chxomato- qsorhy ond paper chromato grophy.2. From o-halogenated acids ox estes :> — The easiest methods for the synthesis of «-amino acids involves the introduction of an amino gsour through nucleophilic displacement in o-haloacids ox theiy esters by Variety of nitrogen nucleophiles. — The w-haloacids or esters are obtained by Hell- Volhor d- Zelinsky ~eaction ox through malonic ester synthesis. — some impostant methods are listed below- @) Divect amination of «- halogenated acids: - Nucleophilic substitution yeaction. af Excess NHs ® e® H,o® NH, + cua So =—aeior H,N— CH,— COONHA ———> H,N— CH.- CooH ca eS Giycme o-haloacid (b> Gabsiel phthelimide synthesis :> -— Phtholimide is a Very geod seagent fox the introduction of a Peimany amino Yeoup into the molecule — This weagent has Widely been used for the synthesis of &- amino acids. 2 CHa ° ats CHs P4@ + EH coasts —> —tn-cooghs _Hyd alysis OF + NHO- by cere ‘ Ge ‘COOK Pot. Salt of o-Bromoestex Phthalimide ‘o Alanine©) Phthalimidomalonic ester Synthesis: > ° COOQH s COOGHS I 24~® + CH coogHe Ss — CH-—CoocHe G ' —Kex > ae ‘© Pot. Salt of Bromomolonic Phthalimidomalonic ester phthalimide oy | corsona ° qneatis COoGHs —Cc¢— coocy .—$—_ a N-—c—cooc.yH 1 — Cele CHRON ° ie > CHS Ces a ° Carbanion Substituted phthalimidomalonic ester “~ a,e® ° ° ° COOH COOH cCHy G He 1 a 1 NH,-NH2 NH 1 —-¢— cooH —> — Ch _ \ + NHS CH-COOH | —COn | NH 0 | «6(Cr Sets 0 | (CHE Ses ‘° Phenylalanine Phtholazine- Altematively , base hyd volysis of phthalimidomalonic estes(a) followed by decasbox ylation gives +the same x&- arwino acid- COOGHS @ Kor, \ a a om — c— coocue @ Dil. HCI Gy &,-Cor CHS gs Substituted phthalimidomalonic ester “@ 3. Streckes Synthesis :> — This Is one of the most convenient method for cH> GH 2 He 1 se = + NHo- CH-COOH ° Phenylalanine Phtholazine Synthesis of 4- amino acids and involves the seaction between an aldehyde , ommoni um chicwage and potassium cyanide. NH4Cl + KCN === NH, + HCN + KCl nH oH Oo ' —H,0 cy, CN) R-¢=0 + NH, —> R- C— Nie eee RCH SE NH Bae \ ow H H Aldi mine H NH2 NH + ( 30 | al R- C-— COOH ——_ R-c-—cN \ H &- amino acid of- amino nitile+. Koop Synthesis : Reductive condensation of «- Keto acids With ammonia: > NH NH2 i HH H2/ Pa , R- Cc eeen & BH an R- ¢— COOH a OR CH— COOH «- Keto acid o-Imino acid &- Amino acd. 5- Schmidt synthesis: so. CH Co — CH-COOGHs «+ HNg __1250 samy Cus ¢— NHK cH-coLgHS 3 R —Na ou \ Hydxazoic ‘ sr Schmidt R Alkyl acetoacetic ester Amino acids :> > Methods of psepovation:» G- Custius teaction.> ee ren liGhgona ° gore Kol (1 mole) P gor NH NH ° gas 4 > zs ae 1S = ih ms cooGhs dy R-* Neeacu oo ~ coogu N CONH-NH 5 hydvolysis s 2 Malonic estes CaHsOH 2e@ 7 COOH Ha,o 5 ae mae « Aor HNO R- cH —_— = ~ = S NHe Hydaolysis NHCOOGHS Y swage : SCcoNs oo amind acid Usethome Pot. salt of malonic acid monoazide — Cyanoacetic estes can also be used jn place ote malonic ester. - N wee UiqHsona a NHNHa geen H-CH —— R- a ene ee R-¢H “ cooghs GW R-* COOGHS \ CONH-NH2 cyanoacetic estes CaHsoH COOH HO,o “ cN Wow ee. cN HNO, R- cH —— k= ——————__ R-&H She Hy dzolysis S Heooghs wwangem Neons o- amines acid7- The Dovapsky synthesis:> ° a : cN u Zon ge Ha /Ni y, R-C-H + H-— CH —— R-CH=C Re cH GH Ncoogts Ncoogug N cooghs Cyanoacetic ester NH na -C,H50H HoOH om ne a po a HNOa x R- cHy- CH <——- _ - ens i sees he : H-C=0 mt ——r [S c- nny jai NeH-NH A \ po =e 1 ye=o ‘se orycoota NH cCo- NH au Co- NH ceaanattihaie Hgdantoin " ScH-NH, | Hot i) | dH COOK Hydavlysis Tsyptophan2: Reduction of oximes and hydrazones of w- Keto acids:> ° NOH 2 u Ht Ml CHy-C—CH- COOGHS + R'oNO ——— > R-C—coogHs + CH,cooH + R'OH a , Alkyl! acetoacetate Alkg! cams nitrite [hatea pie H30r s WH coogi iS ——_— “a s R- CH—COOH Hydzolysis a&-amimo acid NNAG HS > a ® 2 N CHy €— CH= COOGHS + GHe-N=SNCI ———> R=C—CooGHs + CH,COoH R i ‘ Alkyl aceto acetate Diazonium Satt NHa \ R-— CH- COOH «—amino acid FR a es Hydwolysis phenylhydrazone zn/ =<, CH,cooH sts NHa Hot \ R-CH-— COOH.Ie Evlenmeyer azlactone synthesis: > — It is used for synthesis of aromatic amino adds- Gre Hs Gc CH .- NH CeHs Coc CH, — NHCOGHs CoH CHO 1~ i =a | ——_—_—— | Se COOH Nao 4 COOH (h,co),0/ N oO Gilycine Benzaylglycine CHAcooNa * er CHippuric acid) 4 (Hie) Gs Azlactone = C-Cool Tautomerism a . heme c Seer ss Hs CH= ‘ — COOH |e Dil NaOH , Warm (xing NHCOCgHe Lt N=C-oH Gi Hyot Opening) &- Benzoylaminocinnamic acid QWs [rong 4a He-CH,— CH— Cool = He-CHCH— COOH = + CHACOCH Se | He a 7 2 1 NHCOGH. NH2 Phenylalanine? Amine acids: — > Physical propesties of K-amino acids:- — Amino acids axe colourless salt like crystalline solids. ~ They have high melting points and decompose before wetting: — They are soluble in polar salvents but insoluble in Ot ganic solvents. — Theiv aqueous solution exhibit dipole moments. > Chemical reactions :> — Amino acids show three types of chemical weactions- ® Reactions due to amino group. @ Reactions due +o Carboxylic group. © Reactions due to both casboxylic and amino group- ® Reactions due to amino group- 1. Basic natuse:- Se — Arino acids act as Weak bases and hence seact With strong mineral acids to form salts. Nia H3c1e R-CH- COOH + 1 R= cut ée6n By Aikylation : Formation of Betaine:- CHT ® 2cH,t ® RH cH, coo? 3 > CHE NH — CH,Coo® ——S—> (CH,),N — CH, — CooP Betame CNN N- tvimethyl glycine) Zwitterion like character3. Action of nitwous acid «- NH OH 1 \ R- CH —COoH + HNO, ——> R- CH— CooH +N + H20 Hydroxy acid — This weaction fowms a basis of Well-known Van Slyke method for estimation of amino acids. 4. Reaction with nitrosyl chloside o+ bromide:- NH2 cl R- Saecoor + Noc) —— > R- Wy coon + Nz + Hi0 o&- halo acid @ Ss. Acylation: - — Acylamino acids ave obtained Upon treatment with acid chlowideS and anhydrides. (CH3C0),0 + Supers C6oo' ,_—_.. CH,CONH CH, COOH Acetylglycine + Rus CHa coo® ——» GH, CONHCH, COOH Benzoylglycine —These products do not exist in dipolar Porm and ave useful intermediates in peptide synthesis. CeHs COCI6. Action of fosmaldehyde : Fowmel titzation :- @ CHO + NH,— CHz- COOP ——y CH, = NCH,COOH Me thy lenegl yo ne oR ® 2cH,0 + NH;—CH,— coo? ——» (CH,OH),NCH, COOH Dimethyloiglycine — This process is simply @ nucleophilic addition of on amino group to the casbonyl group of Ffosmaldehyde - Wis treatment “ masks” the amino qroup ond hence casboxylic group can be easily treated With alkali (Sovensen fosmol titration). 7 Reaction with Sanger's eagent:- — 2,4-dinitwofluosobenzene (DNFB) is know as Sanger's reagent. Base m+ + Nig cH — COOH —paeee Og HN— CH-CooH HE 0, R ot) e 2 2,4-dinitsophenylamino acid -This teaction is highly useful in detesmining the N-amino acid residue in a peptide ow protein. @ Reactions due te casboxylic_gsoup:> — Amino acids Show acidic character due to presence of a Casboxylic group. This becomes evident by evolution of CO, When on aqueous solution oF amino acid Is treated with NoHCo,.1. Acidic natuse:- — Amino acids seact with base to fowm salts. NH NH : \ e® R-CH- COOH + NaOH ——> R-CH— COONa + H20 2. Estesification: - OH @ Sa cup coo! esa, [ Ru, —cH,-coon | cio = [ NH3- CH coor| cae Ho -H,0 Amino acid Ammo acid ester hydsochlowide hgdrochlovide 3. Fowmation of acid chlowides:- — Conversion of amino acids into corresponding id chlosides is quite difficult because dizect treatment of amino acids with Pcig, SOc, etc: Pails to give the chlovides due to presence of amino group. - To overcome this difficulty, amino adds ove fisst converted into acy! aex CH,- COOH ————> \ NH NHz > lactam — Lactams show a special type of tautomesism called lactam- lactim tautomerism. © R — NH Kia 2 OH Lactam lacknn — If the amino group is psesent farther along the Chain, lineax polymeric amide is the product NH,- (cH) COOH + NH, (cH2),-COOH ——> — NH,ECH,),— CO- Nh} coon Polymeric amide 2. Ninhydvin seaction;- a Ninhgdein weacts with amino acid to give dark blue ox violet colouxed Complex. — This ‘reaction is the basis oF O& Colour test for &~ amino acids- oH Oke +e prem — Ot. Kg R }° Ninhydvin ° © Dosk blue os violet coloured complex3. Formation of hydantoins:- a 9 i els i - =Cc= —_ - = - - ' C$gHg-N=C=O + R cH COOH —> CgHe NH-C-NH-CH-COoH ov o=t c=0 i R Tsocyanate *NH2 . OH NH-C.H 3 Caxrbamide i. ai Carxbarmide R-CH— NH ‘ , | \ HT] Het o= c=0 ~y Hydantoin Hs 4. Formation of metal chelates:— ( — Like other Coxboxylic acids they also fomm metal salts. But their salts qith heavy metals are highly coloured because of the chelate ring sotsucture. Example. Ha co- OH A o=c-0 N—CHa S, : + CuO ———> i can. | CH2-NH2 —H20 H,¢- NX SO—C=0 He Chelate complex (Deep blue)> Peptides:- Peptides ave the polyamides fowmed by +he condensation of amino group of one amino acid With the Carboxylic gsoup of the other. — Secondary amides having —CO-NH-— linkage commonly wefexved as peptide bond ox peptide linkage. NHy— CH= copH | a AEN Gu-cooH 5? Nam CH {co- rn CH— COOH R R R R peptide linkage ox peptide bond A dipeptide > Classification of peptides: > — Depending upon the number of amino acid vesidues per molecule they ave veferred as dipeptides, tvipeptides , tetrapeptides and polypeptides . - proteins are also polyamides - - The compounds having molecular weight of 10,000 os \jess ase called polypeptides while the compounds which have molecular Weight highes than 0,000 ave called proteins. +> Nomenclature of Peptides: — The one end of peptide which have free amino group is called N-terminus amd other end of peptide which have free cavboxylic group is C-texminus. — By convention, peptides ave always Wsitten With N-terminus ow left side and the C-terminus on the wight side.—They ave named from N-texminus to C- terminus through the sequential listin: of the names of amine acids- This 1s dene by weplacing the Ending -ine by —yl_ in the names of all amino acids excepting the c-— tevminus- — The names of polypeptides ave abbreviated by using three letter abbreviation for ayrino adds - Exarn ple => A dipeptide yr teptide linkage A tvipeptide a Oo gnon ® eames ae a —! > ons coo? H3N — CH— feos NH} CH po NH é Gigeylanlonine e Calg » Ala. Sev) R R R @! \ \ a“ H,N- cH — COS NH-CH—COP- NH—CH—Coo! a n > A polypeptide> Geometry of peptide linkage :+ — Studies on Stvucture determination of peptides have ‘*evealed that the amide group is Liat and the cosbony! and amino groups Vie in one plane having H of NH and 0 of Co twans With respect to each other X-Ray studies (done by Linus Pauling) of peptides show +that the C-N bond length of —co-NH— is 132 pm which is shorter than +the usual 147 pm showing Slight double bond character. ° ry 2s" BS) 20" a or 123° tune? ws) oc We. “a ene ON Se Ne \ 123° lor = | H 482 pm a ' ~ Since, Carvbon- nitrogen has so07. double bord character , thevefore the lone paix on the N-atom in the peptide bond is celocalised over c=0 grovp as shown below- “6:2 . N__@ No —_ no = Can ei a H x H Resonating stvuctuse of a peptide bond — Due +o double bond Charactet in C-N bond, the votatio about C- bond is sestvicted . These fore the peptide bond can show geometsical jeomesism.° O° i « i! I cuR' = “Su-N NOu-o=n “aH \ \ \ \ R! H rR! “curR" texans (moxe stoble) crs Cless Stoble) ~ Trans form 1s mose stable than cis form because of much larger Stesic wepulsion between R! and R" GSOUPS- Thus the atoms foxming peptide bond lie in a plane with O and H atoms in trans oxientation. — Freee rotation of a peptide can occurs only avound the bonds joini the nearly Planar amide qsoups to « - carbons. Hence , Confewmation of a protein molecule ox the Polypeptide Chain can be descsibed In terms of angle between R'-CH-NH bends ond +the angle between R'-cH-co bonds. These angles are called Ramchandran angle.(on the name of G-N-A- RamChandtan, Indian bfo- —pysicist) eu Pe XN 7 Planaw peptide bond Fig:- Model of segment of a polypeptide showing planas peptide bonds in boxes and Ramachandran angles of rotation ($ * ¥ ) around a-carbon> Peptide synthesis:> — Synthesis of peptide ig impostant feom two viewpoints — @) The synthesised peptides ave identical with the natural one. @) Synthetic peptides ave useful model compounds for understanding the structural features of proteins - — Peptides ave polyamides and can be synthesised by the stepwise condensation in which the ammo group oF one amino acid is condensed with carboxylic group of a second amino acid: he condensation reaction takes place until a desired polypetide chain is obtained- — The process of peptide synthesis Is complicated because self Condensation of two molecules of same amino adds take place- To overcome this difficulty the ono qrmup or carboxylic groups are proctected oF blocked by conversted into substituted amino or carboxylic qeoup. — The peptide synthesis consists of three steps- fi @) Protection of amino os carborylic group (e) Condensation () Removal of protecting group. (A) Peptide synthesis by protecting arfine group :- @) Protection of amino qroup of amino acidi— — Tis is done by treating the amino acid , which will form N-tewrius of the peptide With a protectn: agent- Tis agent condenses with amino group ef acid and foxsms N- protected atvino acid- [PRJ—x + HyN— CHR— coo” —+ [PR|-NH-cHR- coo Lorotecting qroup N-pwotected amino acid(6) Condensation of the Cosboxylic qroup of the N-pwotected amino add With the aynino qroup of the second amino acid:- [PRI-NH —cHR—COoH + H38 — CHR'— coo® L [PR] — NH- CHR- Co —NH— CHR'— COOH Protected dipeptide This step is *epeated several +ime using desived amino acids one by one till the protected polypeptide of appropriate length is obtained. ©) Removal of the protecting qroup:- iS — The protecting gsoup is vemoved using any usual method taking care to chose such weagents that do not affect the peptide chain. [pr] — NH—- CHR- CO —NH— CHR'— COOH i; 2 1 e [PR]}-H + Hay — CHR — CO— NH— CHR! — CoO OR [pR]-x(8) Ye ide Synthesis by psotectin Casboxylic qsoupi- (@ Protection of the caxboxylic gxoup of amino adid:- — This is done by treating amino acid, which will form the C-terminus of the peptide , with protecting agent -This agent condenses with caxboxylic group of this acid and fosms C-protected amino acid- HeN—cHR— coo® + [PREx ———> h,N— cHR— co —[pR] Protecting guoup C-psotected amino acid ib) Condensation of the Amino qroup of the proteated awino acid with Casboxylic qeoup of the other amino acidt- H.N — CHR! — coo? 4 H,N- CHR -CO-[pR] HN -cHR'_ co-wH- CHR— co—[pR] protected dipeptide Wis step is sepeated ©) Removal of protecting qeoup:- — The protected group is seagents that do not Several me to obtain desixved protected polypeptide - -semoved usin ony of the Usual methods taking cave to choose Such affect the peptide chain. HN -cHR'_ co-wH—- cHR— co—[pR] + H,N -CHR'_ co-WH- CHR— coo? +> The protecting agents: — A number of protecting agents have been developed fos synthesis of peptides. — A good protecting agent 15 that which can veact With the amino or carboxylic easily and can be wemoved , at the end of the synthesis » Without affecting the peptide bond- — Some impoxtant protecting agents are- (A) _N-tewsminus psotecting agents :- Gy Caxbobenzoxy chioside ov Benzyloxycarbony| chlowide. CCgHs CH,0COCI) :- — It is On estes as well as acid chloide of carbonic acid(H,Co,). — I+ Is obtained by treating benzyl alcohol with carbbony| chlovide Cphosgene) . - It ‘weacts with amino compound +o fowm Corresponding substituted amide. This amide on catalytic seduction ox treatment with hydeoloremic ocid in acetic acid, regenerates the amine via casbamic acid - £ Toluene CgHs CHZOH + COC), a CEH CH2Ococl Benzyloxycarbony! chloxide roa | (R> —CHR- Cool) ee wv Her/CHZOH~ Coy CHAO CONH-R 2, fae eeN SERS i. to oa —CO, Cosbamic acid —GHsCHy Substituted amide(i) Phthalic ~amhydaide :+ — The acylation of Free amine group can be done by treating it with phthalic anhydiide- — The wsesulting N- substituted phthalimide on treatment with hydrazine fowms phthalazine and libtates the amme- ° ° NH,- NH + R-NH, ———> Ob —— Pte REND ou ° ° R2 —CHR-COOH N- Substituted phthalimide Phthalazine Git) Aliphatic anhydvides:- — Teifluoroacetic anhydside is used for trifluoroacetylati ng 4+he amino group. — The amide So obtained is converted into amine by treating i+ with ail- NaOH. ° a gR —NH ul i. NaoH Fe - 2-0 -£ -cr, F.C bere ste, | RNS —CF,COOH () C- tewminus protecting agents: - — Estesifaction of camboxylic qroup with benzy! alcohol ts most common method of protecting this qsour- ~The estes is so obtained is converted into the Pree carboxylic acid eithex oy catalytic eduction os hydvolysis of dilute alkali.Hydrolysis RCOOH + Coy CH,-OH ——> = Cg Hy cH,0coR RCooH eeiad -CgHgCH.OH = —CHR-NH2 Ha /Pa Cglig-CH,+ RCOOH> Classical peptide synthesis -+ — Fox the fowmation of a peptide bond, activation of free casboxylic o¥ arnino qeoup of the protected oine acid ox peptide is necessary. This activation can be done by converting the protecting species Into the Corsesponding acid chloride before the condensation step. — Explame:- Synthesis of glycylalanine (dipeptide) G@ Protection of amino group of glycine by treating it with carbobenzoxy chlovide:- CeHg CHO COCL =+ =H,N-CH,—cooH ——> C$eHsCH,OCO — HNCH,COOH Corbobenzoxy chloride Gigcine Cavbobenzoxyglycine &) Activation of carboxylic qroup of carboloenzoxs glycine :- SOCla Og Cg CHOCO — HNCH,COC\ HC! ,-So, oe, Cots CHICO - HNCH,COOH cossesponding acid chlowide ©) Condensation of comcesponding add chliovide, with. alanine TH Sis Ces CH2OC O-HNCH,COCI + HAN —CH- COOH “Sacr els CHO CO-HN cCH{CONH— CH- COOH ) Removal of protecting qvoup Combo benzory glycylolanine : Dee Ha/Pd ng ‘ CeH#gCHOCO-NHCH,CONH- CH-C0eH ET HWCH,CONH — CH- COOH ~ Con Giyeylalani ne Cay -Ale)> Modification of classical peptide synthesis:> — Step &) of the classical peptide Synthesis can be avoided by treating the protected amino add With the second molecule of free amino acid in presence of dicyclo hex! carbodiimide (dec). — Dec isa dely drating agent which is used fox divect conversion of acids inte esters and awides- — Examples synthesis of Alanylglycine @) Protection of amino group of alanine :- chs ens 1 Cele CH-OCOC! + HIN-CH-cCooH =————> CeHgCH,- OCO-HN —CH- CooH protecting agent Alovine Protected alanine We) Condensation of protected omino acid With Qlyiwe In presence of CC. cH3 0 ' ‘“ =) 1 nf Ces CH OCO-HN-CH-CO~G-H+ (_)-N=c=N-{_) + NH CHE COOH —> CgHg CHEOCO-NH- CH —E~ NH-cH coon mee Ggcine protected alanglglycine ©) Removal of protecting gwup:- cH3 0 \ ul CeHs CH,- OCO— NH — CH — C— NH—- CH, — COOH Ho/ Pa 8 CeHs CH, + CO, + HAN-CH—¢ — NH-CH,-COoH Alanylglycine> Solid phase peptide synthesis :> — Classical peptide synthesis has some limitations such as- W Method Work Wel for smaller molecules such as having less than \0 awino acids ‘esidues but laxger molecules axe difficult to synthesise- @) time consuming. WW Pooxer yields - To overcome these limitations, Mewsifield and co-workers (i964) developed a new technique known AS Solid-phase peptide synthesis or Merrifield solid-phase peptide Synthesis OF Modern Ynethod with the help of this method, the enzyme -wibonuclease Ceontai ning 124 amino add yesidues) in time span of only Six Weeks. This is thy this technique is G@lso veferwred to as protein making machine - The rain feature of this technique consist of using insoluble polymer supports Such as halomethy! desivative of polystyrene (Abbteviation PS-cHzx)in presence of Snxq. — Solid-phase peptide synthesis involves Following steps :— di) Synthesis of halomethgl dewivative ‘of polgstiprene:© S CH3OCHAX [Ps]- ch,- x (x= c1,8%) SnXq @) Synthesis of N-protected amino salt :- R R @ | \ OH~ ' - Cos CH,OCOCl + H,N—CH—COO~ ——> CH,CH,OCOCI-NH~ CH-cOOH ———>— CgH CHO CO-NH-CH - C00 Carbobenzoxy amino acid W- protected amino acid N-psotected amino salt chlosideSid Reaction of N-protected amino salt with halomety| devivative of polystyrene = :~ — This teaction involues nucleophilic displacement of halide by Carboxylate ion take place at the benzylic centes of polymer support - * R zx oe ' DMF or 1 I—CH— K + OOCCHNHOCOCH CgH aa — [Ps] ar OOS aa ecsade [B)- cu,— oc cHnHoco cH, ces ¥ DMF > N,N-dimethylformamide N-tevminal protected amino acid desivative dil. HB« / cH,cooh Removal of protecting gvoup ' PB cn,- ooctu wn, 4.00, + cpg cher Polystysene bound amino Qcid derivative Gv) Condensation of polystyrene bound amino acid derivative with N- protected amino acid:- R ’ B)— cxpooce — wn, + HOoccH WHOCOCH Hs * OCC + dicyclchexylcarbodiimide N- protected amino acid | acct catalysed Condensation R ® F ; ' dil- HY . R [Ps] = CH,~ OOCCH NHCOCH NHOCOCH,CQHs = ae? -«u.- OOCCHNHCOCH NH, + CO, + CH CH, By polystyrene bound N-psotected Protecting Polystywene bound dipeptide devivative ee dipeptideiE areaeSEyS of condensation steps :- R R' N-pwotected } amino acid Several steps [PB)- cn. - ooccuwncocH ni, ae — —9 — Be cnpooe------- NHCOOCH, CHE Polystysene bound > Polypeptide chain ‘peptide (Vi) Removal of polystyrene substsate: - ie, [BB)- cH z00¢------- NHcooch cH —H8* _, Polypeptide CFZCOOH Polypeptide chain > Advantages of peptide synthesising ‘machine: 4 — This Solid-phase technique has Now been automated. ~domhputes compatible machines have following advon tage s— @) High yield peptide synthesising fi) Side products can be easily Washed away with negligible loss of the desixed product- Gi Pusification of the products aftex each coupling, Ts not necessary since insoluble polymer ound amino acid or peptide js thowughly washed with Suitable solvents to xemove excess ofthe Beagents. Wy) Since the whole process is automated, it saves a lot of time. y Messifield -cecevied the Nobel Prize for this Wowk.— Peptide stwuctuse determination: > — Structure of peptide can be estabilised by detewmining the number of amino acids constituting the peptide chain and sequence of these amino acids in the peptide chain. 1. Analysis of peptide chain z> = Hydwoly sis of +he peptide with 6N Hel Under nitwogen atomsphere weleases all amino acid except tryptophan which, being a pyrrole, is destroyed by this proceduse. the presence of amino add can be determined by hydvolysis oF polypeptide wrth 2N alkali. However, this procedure , destwoys asginine ‘ castine, sevine and +hreonine and racemises many aMINne acids. vey little ~wacemisation occurs Wy acidic medium. ° o @ uw u e HgN — cH,- C— NH — CMT -Ig0 gC CoS \ cHy Cy Chr CeHs Gia. Ala.Phe [en Hel, No CH, CgH. e ® e @ e ‘Gieaihial CIH,N—CH,— COOH + ClHaNn — fi— coon + CtH,N— CH—COOH CH 3 Giycine hydvochlowide Alanine hydochloside Phenylalanine hydrochlowide - For estimating the amount of each avvine acid from a known weight of the given peptide, Following methods ave Used- ty Ton-exchange chromatography (i) pH dependent precipitation (ii) Electsophowesis2- Sequence of amino acids: End qeoup_analy sis: The sequence of amino acids in a peptide chain is determined by end group analy sis (terminal sesidue oralysis) coupled with partial hydwrolysis. The peptides (and proteins) ore bifunctional molecules having free amino and Carboxylic groups at the end of the chains which axe texmed as N-terminus and C-terminus, Sespectively . These terminals ate detected by using some very specific reagents . When the peptide is treated witha reagent then weagent weacts With a particulay texminus to fowm a tagged peptide. — The tagged peptide weleases tagged amino ocid on partial hydrolysis ( Catalytic ov enzy matis) and tagged amino add is then identified. — The remaining peptide is tagged and subjected to partial hydrolysis again to yield another tagged amino acid Which is again identified. — Wis process is wepeated till all the togged amino acid aye identified, thus, giving an ideaof the amino acid Sequence. — Depending upon the noture of terminus (a) _N-Tesminol sesidue analysis: - rap) Sanges's method: > — The most impostant tagging agent for N-tesminus, is 2,4- dinitrofluoxobenzene CONFB) — DNFB is developed by Frederick Sanger who got Nobel Prize fox detex minating Structuve of Insulin. Insulin is a peptide howmone that controls blood Sugars (evel. , two types of tagging agents have been developed.ee ° ey NO2 ° Oo - \ \ -HF wok pt + H,N-—cH- C-NH- CH- C---- ——> ne{ p-na-« cH-2-Nu- chi= c--- YW a Nucleophilic m substitution & a! Tagged peptide (Yellow) NO2 & a ne{ prnn—cu-E-08 % HN= Chis ¢26Ht.——== etn hydrolysis i * Mixture of untagged Tagged amine acid amino acids CYellou) iy Dansy! method :- > Reagent » sS-dimethylamino-i- ~naphthdlenesulphoryl chloxide — ates os ODNS:Cl) $0,C1 $02-NH— — a NH- ck Ben + HAN- cH - 8 NH- cH geben, oi R Rt Tagged peptide cH Za; 88 DNS.CI arti a cH Neu, Csulp onamide) $0,—-NH—CH- E-0H ° _ yo" “ a aa eo Poxtial hydwolysis Tagges Sele RB! mixture of untagged aN acid + . cH3 CH amino acids— The Dansy! method is better than Sanger's method because W The Covvesponding sulphonariides ore resistant to the action of acids as compare te the dinitvo phenyl substituted amino acids- di) The dansyl group is highly Pluosesent . Thesefose, it allows the estimation of dansy! amino acid even at a verg lower concentsation levels- — Limitation i- — Both the methods descsibed above have a limitation that the hydwolytic cleavage lowing about the complete breakdown of the peptide to the component amino acids, and therefore, the amino acid sequence cannot be easily determined . However _ selective enzymatic ow partial chemical hydvolysis +o smaller fragments Lollowed bey fresh N-terminal tagging ond hychwolys't s has been sucessfully employed for this puspose. = R it) Edman's method: - o \ — Developed by Pehy Edman and modified by several workers. gos Ye P — Reagent > phenylisothiocyanate Cprtc) H—N c , ™ v. ° Noe Nwn-cH-¢-9H- =~ = Gis" N=C=S + HyN-cH-€—NH-CH-C-NH--~ —_, aie a a Sl ip fertic= R -. Tagged peptide Cpeptide substitued \ \ oP +hiourea) cH ° ° cw ‘5 + NZ it he H30* ,»@ ah cH 4 NH-CH-C-NH--- KG ce 3 -4® SZ y S/S GNHECH-C-NH--- ee R i J-e O71 +He Untaggad past of peptide R' C$cHsg-NH having “one amino acid less CgHg-NH Thiolozone thon parant peptide‘ t R \ \ o cH ° cH CS £ a es NF a es Ne ee Zz we -S a Z as SA Ne + es \ e/*x =s 2-H SNH CcHg- NH $ fee s7 See R R el il \ L Se -Hx wf ON ZO H-N / —_— ho=N N \ ef “* ° geek. us — Ne s foe ig. NtgHe Phengithiohydanteim CO) — The above process con be automatically wepeated on the protein molecule Upto Nearly ao amino acid residues. (b> C-Tesminal vesidue analysis :- The chemical method for C—tesminal analysis have not been so sucessful as those used for N-texmmal wesidue onalysis - Two methods Used for this purpose >- dy Enzymatic Method: Selective hydrolysis 0 — Cetyoin enzymes are used fox specific Cleavage of peptides . They ave also peptides but they ave used in Very Small amounts so that they do not inter fere with the peptide omalysis-— Two enzymes, Carvboxypeptidase A and B Cisolated fscm Swine pancreas) are Used fox the catalytic cleavage of only that peptide bond which has a free Carboxylic group at c-terminus. — This give wise +o free amino acid and shostened peptide which has a new C-terminus. The new peptide ts again selectively cleaved +o yield a New amino acid and fuyther Shostened peptide With another new Cc-—+tewminus. This Process wepeats itself step by step till the Whole peptide chain cleaves. — €xample:- In the peptide NH,- A-B-C-D-F-cooK , after ceytain time interval when the peptide is only Pastially hydrolysed , a mixture of amino acids A,B,C,D and E Ts obtained Te welative amounts of these acids Will be ithe osder E>3>C>B>A- he sequence of of amine acids determined on the basis of quant tat ve onaly sis - ii) Hyd azinolysis method:- — In this Method all amino acid except C-tewminus ave converted into hydvazides Ghen treated With hyde azine at 373K. R! R" R" \ | \ ———NH— CHOCO — NHCHCO— NHCHCOOH oe ee? NH.NHa 373k R" a R 1 N N H-CH — CONHNH, + WAN - CH —COOH 1 N,H- CH- CONH NH, Hydvazides Free amino acid— Pzoteins :> — Protein Greek woxd > proteios -> first — Psoteins are complex natural Polymers and ave ated fixst arnong the organic Compounds essential fox gsowth ond maintenance of life - ~— They are present in almost all living cells and found in almost every part of every plant and animal - — In human beings , they awe the moin constituents of muscles, skin hair, nails, tendons,arteries and connecting tissues . — In bsiefly, Each living shell is ade Up of thousand of different proteins. — The proteins present in different plants and animals and even the proteins present in different tissues of a pasticulae part of plant ov animol ate different from one another in the composition and biological functions. (S) > Choractesistics of Proteins: > 1- Elemental composition: > — We proteins are polyamides of &- amino acids- They generally contains C,H,N,0 and s. — The percentage of these elements varies with the source. — The wange of these elements ave- C~> 45-55% H- s-s-9% Oo > 12-307. N— \0-327% S > 0-2-0-37. Sometimes they also contains p,+r and traces of metals such as Fe,Cu,Zn and Mn- 2. High Yoleculay weight :> — They have high molecular weights, genexally above 0,000 and May wun Up to Yillions-BAimphoteric natuve;- — Like amino acids, proteins also have amino and Corboxylic group terminal and ave therefore, 4 amphoteric in natuse- Isoelectsic points:- — The behave as both cations and anion Undes the influence of electric field. Thevefove , they alse Ss: 6- have isolectsic points charactesistic of amino acids- Optical activity:- Au proteins axe optically active because of the presence of chivality centers at o(-position of amino acid wxesicdues- Physical state :- Psoteins ase Colousless and tasteless ‘molecules - They are Soluble in water and their solutions ase colloidal in ‘nature, ‘They can be purified by exystallisation. — Proteins are sensitive +o heat ,acids ,alkalies and mang organic Solvents. 7- Denaturati on ; - When protein is coagulated or precipited by the change in temp of their aqueous solution or addition of acids or alkali then this process js called denaturation of proteins. It is aw iwveversible process. However, in cextoin cases, original propesties of proteins can be sestoed by slow cooling if denaturation has been affected by heat- such process is Called senaturation o% anneoling of the proteins.(8 Colouw veactions:- — Psoteins ase Choracterised easily by the following colour weactions — W Ninhy divin weaction ; - — Wher proteins treated with Pyridine solution of Tinhydein , then they give colours waging fom deep blue to Violet pink, ov even ved in Some cases- di) Biuset test:- — Tis test is chavactesistic of ol) compounds having peptide \inkage ond indicates the preseme of peptide linkage in proteins- — When alkaline solution of protein is treated With a dyop of aqueous copper Sulphate , bluish Voilet colous is obtained - Gi) Millon's ‘eaction:- - — Millon's seagent + HgNo3 In HNO, containing a little HNO, — This test is characteristic of phenols and of only those proteins Which have phenol qvoup ¢-e. those having tyrosine unit- — When such protein is treated With Millon's ‘seagen , Q Ushite precipitate Ts obtained in AS Ginch changes to «ed on heating @v) Kanthoproteic test:- — Protein upon treatment With itsic acid give Yellow os orange Colous. This is why our skin turns getlow When it comes in Contact With nitric acia- ©) Helles's test:— — When conc. HNo; is poured along the sides of a test tube containing protein solution ,a ppt is appears at the junction of two layers. This test is commonly used for detecting albumin in urine.> Classification of proteins:> (A) Classification on the basis of their stwuctuves and features :—- W Fibyous proteins3> — They ore threadlike shape and fosm fibres. — They ave genewally insoluble in watex and other common solvents but 3 4 alkaline solutions. — @g- (a) Kesatin — found in hair, Skin, noils etc- (b) Myosin — a protein Foxrring musculae fibre- (i) Globulas proteins:7 — They ave sphexical is shape. - They are Soluble in water and dilute acids ov alkalies. — @g- @) Twsulin - found in pancreas &) Albumin - gq white @) Classification on the basis of hydwolysis products: - G) Simple proteins: - soluble in Strong acid oF — They are simple polyamides Which give only amino adds on hydvoly sis- @) Aibumins Clwater Soluble proteins,eg-, 9a albumin , Sesum albumin etc-) (b) Gilobulins ( Woter insoluble but Soluble in ailute Salts, acids or alkali,e-g- sexsum globulin, tissue globuline etc)©) Glutelins CInsoluble in wates ox Salt Solution but soluble in dilute alkalies oO acidS,e.g-, gluetin from wheat and oxyzexin from wice) Gd) Albuminoids oF sclesoproteins (Wwates insoluble but soluble in strong acid o% alkali solutions ,e-g) Kesatin tibsorim etc. (i) Conjugated proteins:+ — They ase psoteins Which have some yon-protein vesidue attached to the protein molecules- — Such Non- proteins moieties ore called the psasthetic groups. — The main functions of prosthetic group is to control the specific biological action of pyoteins- — They are sudivided into Fforlowing — @) Chromoproteins Chaving coloured prosthetic qsoup ,e-g., haemoglobin has porphin nucleus as the prosthetic group) Nucleoproteins Cthey contain nucleic acid as prosthetic group) ©) Glycoproteins (+hey contain sugar wesidue as prosthetic group) @) Phosphoproteins ( they have phosphoric acid vesidue as prosthetic gxoup) ©) Lipoproteins Gi) Dewvived proteins :> — These ate lowes proteins or peptides and ave formed by degradation of proteins os enzymes. — @g. denaturated proteins, proteoses , peptones and polypeptides Proteins ——> Proteoses —> Peptones —> Polypeptides— Stwuctuve of proteins and levels of pxotein stwuctuse:- — Styuctuse ond shape of protems can be studied at fous different levels :- G) Primary structure: + — Primoxy stvuctuve of proteins contain one or more Polypeptide chains and each polypeptide has amino acids linked with each othes in a specific sequence. This Sequence iS called primary stvuctuse of protein. v— Peptide linkage a 1L@-_ i i — “In primary structure of proteins every thisd atom of the peptide chain contains a group xeferved 4o aside chain? which is characteristic of the amino acid present. exomple -H > Gigcine —-CHICgHs > Phenylalanine —NH2> Lycine —cocn — Aspastic acid — ‘The difference in number of acidic or basic side chains is responsible for separation of proteins by electyop hoxesis - — The procedure for determination of numbex of amino acid molecules and its sequence 1S Same as peptides. — Any change in the sequence of amino acids Creates a different proteins.Q) Secondary structure i> — In these type of structures, peptide backbone can intewact itself . — These interactions depend upon size of the ‘side chain’. — Depending upon the size of group R, following different steuctures ase possible- @) Flat sheet:> — If the case of proteins having side chains of smaller size (say H), the peptide Chains ave segarded as futly extended in zig-zag manner With alternate side chains being on the same Side situated at a fixed distance. — Such extended peptide chains lie side by side and each ove is joined to two neighbouring chains +hrough hgdsogen lbonds. This results In the fowmation of flat sheet. oO H Oo H I ee | c N ££ \ aN 7 Ze \cZ S \v” x a ‘a fav il | ~ Il Repitition 0 Hy Q <=> of similar +—+ E< Hydaogen bond ——> structural : 5 3 : H oO H units | I «7 | = VPA LV AS AS ¢ OC Nc J 2N I t Figi- Flat sheet secondasy structure of peotein(b) Pleated sheet +5 — If the side chain is of modewate size , the peptide chain contracts a littile in order to accommoedat im the pleated sheet stsucture ov the B- awrangement of the protein. hydrogen bonds- them. This wesults - The distance between alternate chains becomes shost and ‘hey ore held together bg The B-pleated sheet of protem Fig:- ©) o- Helical stsuctuse:> — If the side chains awe quite large, then whole peptide chain is coiled in pasticulos helical form Known as o-helix- — As all the Naturally occusing amino acids have L- configuration , oll protein helices have wight handed-— Each omide group is hydrogen bonded With every thivd amide in either disection along the chain. -— 3:6 amino acid units form one turn of helix and the Side chains extend out away feom the axis of this helix- Fiqi- A right handed «- helical stxucturce et ee: typical psotein (3) Textiary Ssteuctuse.> — The tertiory structure of protein constitutes a three-dimencional shape due to further folding of polypeptide chain having Compact » highly Complex gteucture Which is characteristic of a pita Protein.— Textiory structures of protein contain Follow ing bonding Interaction between the amino acid chains- (@) Very weak Van der Waals foxcos+ €-g- intevaction between tryptophan and phenylalanine sesidues . (b) Additional hydsogen bonds— Apart From Usual ones . €-g: hydsogen bonding between side chains of sevine and histidine. €) Intvamolecular salt like dipolae bonding > €.g- between ‘extra’ Carboxylic and amino groups of aspastic acid and lysine, wespectively- @) Disulphide covalent bending> e-g- between tudo cysteine wesidues . —The tertiary structure folds the entire protein molecule in a pazticular shape and Stablises the pwotein roolecule- Mis pazticulay type of Folding is called native conformation of protein. — Globulas and Fibrous shape ave tertiary stwuctures of proteins: Globular proteins ave Sphesical in shape and Fibrous proteins have ‘wod-like Shape. Howe unit @) Quotesnary stwuctuse: > — Some of the proteins ave composed of two or more Polypeptide chains. These axe called cub-units- — The spatial arsangement of these subunits with respect to each othes IS Knoton as quaternary Stwucture. — Example :- Myoglobin Ccontain haeme unit as Prosthetic gxoup) Figi- Diagramatic ~representation of Myoglobin molecule.> Trathine _danotuwatton/venabenation:y — Proteins ave sensitive to heat, mineral acids, alkalies etc. — When soluble form of proteins ore heated or treated with mineval acids, they undergo coaguation ov precipitation to given fibsous Protein hich ave insoluble in Water. The coagulated protein axe Called denatuved proteins ond this process is called denatusation of pvoteing- This coagulation also wesults in the loss of the biological activity of the psoteins. Chemically , denaturation dees Not change the primasy Stvuctuse but brings about change in the secondary and tex tiasy Steuctuses of proteins. Random coil — aes © Native protein Denaturated protein Fig:- Denaturation of globulas protein - Examples of denaturation of proteins ave:- @) Coagulation of albumin present in €9g White :- — The soluble globular pxotein (albumin) present in the egq is denatuved when the egg is boiled howd wesulting in the fosmation of insoluble fibrous psoteins.(b) Coagulation of milk:- — When milk is heated With an acid (lemon juice or tartaric acid) the denaturation of milk leads to fowmation of cheese, Dusing this denaturation, the globular milk protein ( lactalbumin) becomes fibvous- — Eavlies, denatusation, was considered to be ixseversible Process. However, Now it has been Shown that in Some cases, the pwocess is actually seversible . The -reverse process is called wenaturation Example:- When temp- and pH of denatured protein are bought hacte to Conditions Which Stabilize Native protein, secon day and textiony stwictures of the proteins ave yestowed. consequently , senaturation is also accompanied by wecovery of the biological activity particulary in case of enzymes .