Proteins

AMINO ACIDS

Introduction

(i) Amino acids are organic compounds of both, an amino group & carboxylic group.

(ii) They are represented by general formula:

(iii) These amino acids are very important because they are the building blocks of protein.

(iv) Protein is the natural polymer moving $\alpha$ - amino acids as monomer.

(v) With the exceptions of glycine. All the other amino acids have chiral carbon & have two optically active isomers.

(vi) All naturally occurring amino acids are in L – series in which $-$ NH₂ group on the left and $-$ OH group in the right as L – glyceraldehydes.

Illustration 1. Which among the following statements are true for glycine?

1. It exists in crystalline form 2. It is optically active

3. It is soluble in water 4. It can form Zwitter ions

(A) 1, 2 and 3 (B) 1, 2 and 4

(C) 1, 3 and 4 (D) 2, 3 and 4

Solution: (C) It is ionic so it exists in crystalline form. Because it is ionic so it soluble in water. Because acidic and basic groups are present in the same molecule, so internal salt formation occurs to form dipolar ion NH₃⁺ $-$ CH₂ $-$COO^-

Classification

Amino acid with non – polar side chain

Essential amino acids

(a) These must be supplied to our diet as are not synthesized in body.

(b) Some of them are

(1) Valine (2) Leucine (3) Isoelucine (4) Phenylalanine (5) Arganine (5) Threonine

(6) Tryptophan (7) Methionine (8) Lysine (9) Arginine (10) Histadine

Note: Histidine and arginine are essential i.e. can be syntrhesized but not in quantities sufficient to permit normal growth.

Non – Essential Amino acids

These amino acids are synthesized in body.

Some of them are

These are as follows:

(1) Glycine (2) Alanine (3) Tyrosine (4) Serine (5) Cystine (6) Proline (7) Hydroxyprocine

(8) Cysteine (9) Aspartic acid (10) Glutonic acid

Synthesis of $\alpha$ - amino acids

Protein can be hydrolyzed by refluxing with dilute hydrochloric acid to give a mixture of $\alpha$ - amino acids. The resulting mixture can be separated by

(a) fractional crystallization.

(b) Fractional distillation of their ester followed by hydrolysis (Fischer's method)

(c) Selective precipitation as salt with phosphotungstic and picric acid.

(d) Distribution of amino acid between n – butanol saturated with water (Dakin's method).

(e) Column, paper and gas chromatography.

(f) Electrophoresis.

By amination of $\alpha$ - halo acid

(i)

$\underset{Chloroaceticacid\left(excess\right)}{Cl-C{H}_2-COOH+3N{H}_3}\underset{\left(-N{H}_{4}Cl\right)}{\overset{{50}^{0}C}{\to }}{H}_{2}N-C{H}_2-CO{O}^{-}N{H_4}^{+}\stackrel{H^{+}/H_{2}O}{\to }\underset{Glycine}{H_{2}N-C{H}_2-COOH}$

(ii)

By Gabriel synthesis

By Strecker Synthesis

Note: Generally the aldehyde is treated with a mixture of ammonium chloride and potassium cyanide in aqueous solution.

$N{H}_{4}Cl+KCN\stackrel{Aqueous}{\to }N{H}_{4}CN+KCl$

$N{H}_{4}CN\stackrel{Aqueous}{\to }N{H}_3+HCN$

Illustration 2.

Compound X, Y, Z are

(D) All are correct

Solution: (C)

Physical properties of amino acid

(i) Amino acids are generally, crystalline substance having sweet taste.

(ii) They melt with decomposition at higher temperature (more than 200°C).

(iii) They are soluble in water but insoluble in organic solvents.

ZWITTER ION

(i) Amino acids contain both acidic carboxyl group (-COOH) and basic amino group in the same molecules.

(ii) In aqueous solution, the acidic carboxyl group can lose a proton and basic amino group can gain a proton in a kind of internal acid – base reaction.

iii) The product of this internal reaction is called a Dipolar or a Zwitter ion.

(iv) The Zwitter ion is dipolar, changed but overall electrically neutral and contain both a positive and negative charge.

(v) Amino acid in the dipolar ion form are amphoteric in nature.

(vi) Depending upon the pH of the solution, the amino acid can donate or accept proton.

Illustration 3. In acidic medium, amino acids are present as

Solution: (C)

ISOELECTRIC POINT

(i) When ionized form of amino acid is placed in an electric field it will migrate towards the opposite electrode.

(ii) Depending upon the pH of the medium following three thing may happen.

(a) In acidic medium, the cation move towards cathode.

(b) In basic medium, the anion move towards anode.

(c) The Zwitter ion does not move towards any of the electrodes.

(iii) At a certain pH (i.e. H⁺ concentration), the amino acid molecules show no tendency to migrate towards any of the electrodes and exists as a neutral dipolar ion, when placed in electric field is known as isoelectric point.

(iv) All amino acids do not have the same isoelectric point & it depends upon the nature of

R – linked to $\alpha$- carbon atom.

Illustration 4. Pick out incorrect statement

(A) In an electrolysis experiment, $\alpha$ - amino acids do not towards any of the electrode at the isoelectric point.

(B) p-aminobenzenesulphonic acid is a dipolar ion; while p-aminobenzoic acid does not

(C) Sulphanilic acid is soluble in base, but not in acid

(D) H₃N⁺CH₂COOH (pka = 2.4) is more acidic than RCH₂COOH (pK_a = 4 – 5)

Solution: (A) The pH at which |Anion| = |Cation| is called isoelectric point. At isolectric point, $\alpha$ - amino acids to not migrate when field is applied.

(B) -SO₃H is strongly acidic and donates H⁺ to weakly basic arylamino group. ArCOOH is not acidic enough to transfer H⁺ to the arylamino group.

(C) In p- H₃N⁺ C₆H₄SO₃^-, H₃N⁺ is acidic enough to transfer H⁺ to bases to give the soluble anion p – H₂N – C₆H₄SO₃^- – SO₃ is too feebly basic and cannot accept H⁺ from acids.

(D) $\alpha$ - H₂N⁺ group increases acidity, because of its electron – withdrawing inductive effect.

Hence (D) is correct.

Isoelectric point of some amino acid

(v) Amino acids have minimum aqueous solubility at isoelectric point.

Chemical Properties

Amino acids show the following characteristic reactions.

1.Reaction of the carboxyl group.

2.Reaction of the amino group.

3.Reaction involving both the carboxyl and the amino group.

Reaction of the carboxyl group

Reaction with base

(i)

$\underset{Glycine}{H_3{N}^{+}-C{H}_2-CO{O}^{-}}+NaOH\to \underset{Sodiuma\min oacetate}{H_{2}N-C{H}_2-CO{O}^{-}N{a}^{+}}+H_{2}O$

Mechanism:

Esterification

$\begin{gathered} \mathop {{H_3}{N^ + } - C{H_2} - CO{O^ - }}\limits_{Glycine} \xrightarrow[{}]{{HCl}}C{l^ - }\mathop {{H_3}{N^ + } - C{H_2} - COOH}\limits_{} \xrightarrow[{}]{{{C_2}{H_5}OH}}C{l^ - }{H_3}{N^ + } - C{H_2} - COO{C_2}{H_5} \hfill \\ \,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\,\xrightarrow[{}]{{AgOH}}\mathop {{H_2}N - C{H_{2}} - COO{C_2}{H_5}}\limits_{Ethyl - \alpha - a\min o\,\,acetate} + AgCl + {H_2}O \hfill \\ \end{gathered} $

Note: HCl first converts the dipolar ion into an acid which is subsequently esterified.

Decarboxylation

$\underset{Glycine}{H_{2}N-C{H}_2-COOH}+Ba{\left(OH\right)}_2\stackrel{\Delta }{\to }\underset{Methyla\min e}{C{H}_3-N{H}_2}+BaC{O}_3+H_{2}O$

Reduction

Reaction with strong acid

$\underset{Glycine}{H_3{N}^{+}-C{H}_2-CO{O}^{-}}+HCl\to \underset{Glycinehydrochloride}{C{l}^{-}{H}_3{N}^{+}-C{H}_2}-COOH$

Acetylation

Reaction with Nitrous acid

$\underset{Glycine}{H_3{N}^{+}-C{H}_2-CO{O}^{-}}+\underset{Nitrousacid}{\mathrm{HONO}}\to \underset{Glycollicacid}{HO-C{H}_2-COOH}+N_2\uparrow +H_{2}O$

Note: (i) This reaction forms the basis of the "van slyke method" for the estimation of amino acids.

(ii) The nitrogen is evolved (one half comes from the amino acid) quantitatively and its volume measured.

Reaction with Nitrosyl halide

$\underset{Glycine}{H_{2}N-C{H}_2-COOH}+NOCl\to \underset{Chloroaceticacid}{Cl-C{H}_2-COOH}+N_2\uparrow +H_{2}O$

Reaction with 2, 4 – Dintrofluorobenzene (DNFB)

Reaction involving both the carboxyl & the amino group

Effect of heat

$\alpha$ - amino acids undergo dehydration on heating (200°C) to give diketo piperazines.

PEPTIDES & PROTEINS

Introduction

(i) Proteins are formed by joining the carboxyl group of one amino to the $\alpha$ - amino group of another acid.

(ii) The bond formed between two amino acids by the elimination of water molecules is called peptide linkage.

(iii) The product formed by linking amino acid molecules through peptide linkage $-$CO $-$ NH $-$ is called a peptite.

(iv) When two amino acids combined in this way the resulting product is called a dipeptide.

(v) Peptide are further designated as tri, tetra or penta peptides accordingly as they contain three, four or five amino acid molecules, same or different.

(vi) In a peptide the amino acid that contains the free amino group is called the N – terminal residue (written on L.H.S).

(vii) The amino acid that contains the free carboxyl group is called the C – terminal residue (written on R.H.S).

(viii) If a large number of $\alpha$ - amino acids (100 to 1000) are joined by peptide bonds the resulting polyamide is called polypeptide.

(ix) By convention a peptide having molecular weight upto 10,000 is called polypeptide.

(x) While a peptide having a molecular mass more than 10,000 is called a protein. '

Structure of Proteins

(i) Proteins have three dimensional structures.

(ii) There are number of factors which determine the exact shape of proteins.

Structure of Proteins

Primary structure

(i) This type of structure was given by Friedrich Sanger in 1953 in Insulin.

(ii) Primary structure is conformed by single polypeptide chain in a linear manner.

(iii) All amino acid are attached in a straight chain by peptide bond.

Secondary structure

(i) The fixed configuration of polypeptide skeleton is referred to as the secondary structure of protein.

(ii) It gives information

(a) About the manner in which the protein chain is folded and bent.

(b) About the nature of the bond which stabilizes this structure.

(iii) This structure of protein is mainly of two types

(A) $\alpha$-Helix

(a) The chain of $\alpha$-amino acids coiled as a right handed screw (called $\alpha$-helix) because of the formation of hydrogen bond.

(b) The spiral is held together by H-bonds between N–H and C = O group vertically adjacent to one another.

(c) X-Ray studies have shown that there are approximately 3.6 amino acid unit for each turn in helix.

(d) Such proteins are elastic i.e., they can be stretched.

(e) On stretching weak H-bonds break up and the peptide act like a spring.

(f) The hydrogen bonds are reformed on releasing the tension.

e.g. Myosin, Keratin, Tropomysin.

(B) Beta-pleated sheet

(a) Polypeptide chains are arranged side by side.

(b) The chains are held together by a very large number of hydrogen bond between

C = O and NH of different chains.

(c) These sheets can slide over each other to form a three dimensional structure called a beta pleated sheet.

e.g. Silk has a beta pleated structure.

Tertiary structure

(i) It refers to the arrangement and interrelationship of the twisted chain of protein into specific layer or fibres.

(ii) This tertiary structure is maintained by weak interatomic force such as, H-bonds hydrophobic bond, van der Waals' force and disulphide bonds (eg Insulin).

e.g. Protein of tobacco mosaic virus (TMV); Myoglobin; Hemoglobin

Quarternary structure

(i) When two or more polypeptide chain united by the force other than covalent bond i.e., peptide and disulphide bonds.

(ii) It refers to final three dimensional shape that results from twisting bonding and folding of the protein helix.

(iii) It is most stable structure.

Classification of Proteins

There are two methods for classifying proteins.

(i) Classification according to Composition

(ii) Classification according to Functions

Illustration 5. Secondary structure of proteins is due to

(A) peptide bond (B) hydrogen bond

(C) covalent bond (D) co-ordinate bond

Solution: Hydrogen bond.

Classification according to Composition

Simple proteins

(i) Simple proteins are those which yield only $\alpha$-amino acids upon hydrolysis.

(ii) Simple proteins are composed of chain of amino acid unit only joined by peptide linkage.

Examples are:

Egg (albumin); Serum (globulins); Wheat (Glutelin); Rice (Coryzenin)

Conjugated proteins

(i) Conjugated proteins are those which yield $\alpha$ - amino acids plus a non protein material on hydrolysis.

(ii) The non protein material is called the prosthetic group.

Example:

Casein in milk (prosthetic group is phosphoric acid); Hemoglobin (prosthetic group is Nucleic acid); Chlolesterol (prosthetic group – lipid).

According to molecular shape, proteins are further classified into two types.

(A) Fibrous protein

(a) These are made up of polypeptide chain that are parallel to the axis & are held together by strong hydrogen and disulphide bonds.

(b) They can be stretched & contracted like thread.

(c) They are usually insoluble in water.

Example:

Keratin (hair, wool, silk & nails); Myosin (muscle)

(B) Globular Proteins

(a) These have more or less spherical shape (compact structure).

(b) $\alpha$ - amino helix are tightly held bonding; H – bonds, disulphide bridges, ionic or salt bridges:

Examples:

Albumin (egg)

Classification According to functions

The functional classification includes following groups.

Structural proteins

These are the fibrous proteins such as collogen (skin, cartilage & bones) which hold living system together.

Blood proteins

(i) The major proteins constituent of the blood are albumin hemoglobin & fibrinogen.

(ii) Their presence contribute to maintenance of osmotic pressure, oxygen transport system & blood coagulation respectively.

Tests of protein

Biuret test

(i) On adding a dilute of copper sulphite to alkaline solution of protein, a violet colour is developed.

(ii) This test is due to the presence of peptide $\left(-\stackrel{\stackrel{O}{||}}{C}-NH-\right)$ linkage.

Millon's test

(a) Millon's reagent consists of mercury dissolved in nitric acid (forming a mixture of mercuric & mercurous nitrates).

(b) When millon's reagent is added to a protein, a white ppt is formed, which turn brick red on heating.

(c) This test is given by protein which yield tyrosine on hydrolysis (due to the presence of phenolic group).

Nihydrin test

(i) This test is given by all proteins.

(ii) When protein is boiled with a dilute solution of ninhydrin, a violet colour is produced.

Uses of Proteins

(i) Protein constitute as essential part of our food, meat, eggs, fish, cheese provide protein to human beings.

(ii) Casein (a milk protein) is used in the manufacture of artificial wool & silk.

(iii) Amino acid needed for medicinal use & feeding experiment, are prepared by hydrolysis of proteins.

(iv) Gelatin is used in desserts, salad's, candies bakery goods etc.

(v) Leather is obtained by tanning the protein of animal hides.

(vi) Hemoglobin present in blood is responsible for carrying oxygen and CO₂.

(vii) Hormones control various process.

(viii) Enzymes are the proteins produces by living system & catalyse specific biological reaction.

Example:

Ureases (Urea $\to$ CO₂ + NH₂)

Pepsin (Protein $\to$ Amino acid)

Trypsin (Protein $\to$ Amino acid)

Carbonic anhydride (H₂CO₃ $\to$ H₂O + CO₂)

Nuclease (RNA, DNA $\to$ Nucleotides)

Isoelectric Point

What happens when a solution of an amino acid is placed in an electric field depends upon the acidity or basicity of solution. In quite alkaline solution.

Anions (II) exceed cations (III), and there is a net migration of amino acid toward the anode. In quite acidic solution cations (III) are in excess, and there is a net migration of amino acid towards the cathode. If (II) and (III) are exactly balanced, there is no net migration; under such conditions any one molecule exists as a positive ion and as a negative ion for exactly the same amount of time and any small movement in the direction of one electrode is subsequently cancelled by an equal movement back towards the other electrode. The hydrogen ion concentration of the solution in which a particular amino acid does not migrate under the influence of an electric field is called the isoelectric point of that amino acid.

An amino acid shows its lowest solubility in a solution at the isoelectric point, since here there is the highest concentration of the dipolar ion. As the solution is made more alkaline or more acidic, the concentration of one of the more soluble ions, II or III increases.

If an amino acid has amino group and one carboxyl group, it has two pK values. The isoelectric point (PI) of this amino acid has the average value of the both pK values.

We take example of glycine.

H₃⁺N $-$ CH₂ $-$COOH $\rightleftharpoons$ H₃N⁺$-$ CH₂$-$ COO^– + H⁺ …(1)

Conjugated acid (CA) Dipolar Ion (DI)

At equilibrium$K_1=\frac{[DI][H^{+}]}{[CA]}$

H₃N⁺$-$ CH₂ $-$ COO^– $\rightleftharpoons$H₂N $-$ CH₂ $-$ COO^– + H⁺ …(2)

DI Conjugated Base (CB)

At equilibrium $K_2=\frac{[CB][H^{+}]}{[DI]}$

[CA] = $\frac{[DI][H^{+}]}{K_1}$

[CB] = $\frac{K_2[DI]}{[H^{+}]}$

At isoelectric point [CA] = [CB]

$\frac{[DI][[H_i^{+}]}{K_1}=\frac{K_2[DI]}{[H_i^{+}]}$ Where $[H_i^{+}]$ = conc. of [H⁺] at isoelectric point.

or, ${[H_i^{+}]}^2$= K₁ K₂

or, 2log [H_i⁺] = log K₁ + log K₂

or –2 log (H_i⁺] = - log k₁ – logK₂

or 2pH_i = pK₁ + pK₂

or pH_i = $\frac{p{K}_1+p{K}_2}{2}$