© E.V. Blackburn, 2012 Amino Acids and Proteins. © E.V. Blackburn, 2012 The hydrolysis of most...

© E.V. Blackburn, 2012

Amino Acids and Proteins


The hydrolysis of most proteins produces about twenty different amino acids.

an -amino acid

C COH

OH

NH2

R

Structure of amino acids

The acids have an amino group bonded to the carbon:-


Amino acid classification

• neutral - one amino and one carboxyl group

• acidic - one amino and two carboxyl groups

• basic - two amino and one carboxyl group

The 20 amino acids are further classified:


Neutral amino acidsName Symbol Structure

Alanine Ala or A CH3CH(NH2)CO2HAsparagine Asn or N H2NCOCH2CH(NH2)CO2HCysteine Cys or C HSCH2CH(NH2)CO2HGlutamine Gln or Q H2NCOCH2CH2CH(NH2)CO2HGlycine Gly or G CH2(NH2)CO2HIsoleucine Ile or I CH3CH2CH(CH3)CH(NH2)CO2HLeucine Leu or L (CH3)2CHCH2CH(NH2)CO2HMethionine Met or M CH3SCH2CH2CH(NH2)CO2HPhenylalanine Phe or F C6H5CH2CH(NH2)CO2HProline Pro or P N CO2H

H


Name Symbol Structure

Threonine Thr or T HOCH(CH3)CH(NH2)CO2H

Tryptophane Try or W NH

CH2CH(NH2)CO2H

Neutral amino acids

Serine Ser or S HOCH2CH(NH2)CO2H

Valine Val or V (CH3)2CHCH(NH2)CO2H

CH2CH(NH2)CO2HHOTyrosine Tyr or Y


Aspartic acid Asp or D HO2CCH2CH(NH2)CO2H

Acidic amino acidName Symbol Structure

Glutamic acid Glu or E HO2CCH2CH2CH(NH2)CO2H


Arginine Arg or R HN=C-H(CH2)3CH(NH2)CO2H NH2

N NH

CH2CH(NH2)CO2HHistidine His or H

Lysine Lys or K H2N(CH2)4CH(NH2)CO2H

Name Symbol Structure

Basic amino acids


Essential amino acids

Humans can synthesize only 10 of these amino acids. The remaining amino acids must be obtained from dietary sources and are so called essential amino acids.

These are: Isoleucine, Leucine, Methionine, Phenylalanine, Threonine, Tryptophan, Valine, Arginine, Histidine, and Lysine.


Stereochemistry of -Amino Acids

Most naturally occurring amino acids have the same configuration about the carbon:

CHO

CH2OHHO H

CO2H

CH2OHH2N H

CO2H

RH2N H

L-(-)-glyceraldehyde L-(-)-serine natural amino acids


Acid-base Properties of Amino Acids

RCHCO2H

NH2

-amino acid

RCHCO2-

NH3+zwitterion

H+

OH-

RCHCO2H

NH3+

OH-H+

RCHCO2-

NH2


Physical Properties

• very water soluble

• insoluble in non-polar organic solvents such as ether

• high melting points with decomposition


Isoelectric Points

- the pH of a dilute aqueous solution of the amino acid at which the amino acid is exactly neutral.

The isoelectric point of neutral amino acids lies between pH = 4.8 and 6.3. For the basic amino acids, it is in the range 7.8 to 10.8. The range is 2.7 to 3.2 for acidic amino acids.


RCHCO2H

NH3+

At a pH above the isoelectric point, amino acids form anions; at a lower pH, protonation occurs and cations form. An amino acid is the least soluble at its isoelectric point.

RCHCO2H

NH2

-amino acid

RCHCO2-

NH3+zwitterion

OH-H+

H+

OH-

RCHCO2-

NH2

Isoelectric Points


Electrophoresis

• The amino acid is spotted on a strip of paper (or gel) on a center line.

• The strip is moistened with a buffer solution of a given pH.

• Electrodes are attached to the ends of the slip and a potential applied.

• Depending on the amino acid’s charge, it will migrate either to the positive or negative electrode. The rate and direction of migration depends on the buffer pH and the isoelectric point of the amino acid.

Electrophoresis uses isoelectric point differences to separate amino acids and proteins.


Synthesis of amino acids - the Hell-Volhard-Zelinsky reaction

CH3CH2CO2H1. Br2/PBr3

2. H2OCH3CHBrCO2H

excess NH3

CH3CHCO2HNH2

alanine


Synthesis of amino acids - the Strecker synthesis

NH3 adds first to give an imine then CN- attacks

NH4Cl/KCN

H2O

RCHCO2HNH2

CHO

R C NRHCNH2

H3O+


The Peptide Bond

CR

HCO

NH

CR

HCO

N CH

R

H

The principal chain contains the amide bonds; the substituents, R, make up the side chains. The individual amino acids which make up the peptide are often referred to as amino acid residues.

In some proteins, two or more polypeptide chains are joined together by disulfide bridges.


NomenclatureStarting from the free amine end of the peptide, the names of the amino acids are joined together with each amino acid being considered as a substituent of the following amino acid. The name ends with the name of the final amino acid:

H3NCH+

C6H5CH2

CO

NHCHCH2

(CH3)2CH

CO

NHCHCO 2-

HCOHCH3

phenylalanylleucylthreonine

Phe-Leu-Thr


Aspartame

• an artificial sweetener sold under the trade name Nutrasweet

aspartylphenylalanine methyl ester

Asp-Phe-OCH3

• the ester ending is indicated by -OCH3 in the abridged notation.

H3N-CH-C-NH-CH-C-OCH3

O O

CH2

CO2-

CH2-C6H5

+


Angiotensin II

Angiotensin II is a blood pressure regulating hormone. It contains 8 amino acid residues. It is possible to arrange these in 40,320 different ways only one of which corresponds to the hormone!

Its structure is actually: Asp-Arg-Val-Tyr-Ile-His-Pro-Phe.


Sequential Degradation of Peptides

Step 1: breaking disulfide linkages and purification of products

Many polypeptides consist of two or more chains which are joined together by disulfide bridges. These bridges must be broken and the fragment products separated. This is done by an oxidation:

S SHCOOH

O

SO3H HO3S

chain A chain B chain A chain B


Purification

The various methods used to separate polypeptides depend on size, solubility in a given solvent, charge or ability to become bonded to a support material (chromatography).

• dialysis - filtration through a semi-permeable membrane

• ion exchange chromatography

• electrophoresis

• chromatography


The polypeptide is completely hydrolyzed (HCl 6M, 110C, 24hr) to give a mixture of the free amino acids:

H3N-CH-C-NH-CH-C-OH

O O

CH3 CH(CH3)2

Ala-Val

H2O

HCl 6M

110o, 22hr

+

H3N-CH-C-OH

O

CH3

Ala

+ H3N-CH-C-OH

O

CH(CH3)2

Val

+ +

Step 2. What amino acids are present?


Amino Acid AnalysisThe mixture is separated using an automated system.

The apparatus includes an ion exchange column with a negatively charged support (usually carboxylate or sulfonate). The amino acid mixture, in weakly acidic solution, is added to the column.

According to structure, the amino acids are more or less protonated and so will be retained more or less strongly on the column. The pH of the eluant is gradually increased which results in deprotonation and the subsequent elution of the amino acids.

Elution begins with the strongest acid and ends with the weakest.


Amino Acid AnalysisThe eluant then passes into an analyzer which holds a special indicator, ninhydrin. The amino acid eluant forms a violet-purple color by reaction with the ninhydrin and the color’s intensity is proportional to the quantity of acid present.

pH

Asp

Thr

Ser Glu

Pro

Gly Ala


There are a number of methods used to determine the N-terminal amino acid of a peptide. They are all based on the fact that it is the only amino acid with a free amino group.

Step 3. The sequential analysis of amino acids starting at the free

amino end


Sanger Degradation

FNO2

H2NCH2CONH

NO2

NHCH2CONHNO2

NO2


Edman DegradationPhenyl isothiocyanate, C6H5N=C=S, is a reagent which permits the progressive removal and identification of the N-terminal amino acid. The rest of the chain is not affected by the reaction therefore progressive removal is possible.


Edman Degradation

Ph-N=C=S + H2N-CH-C-NH-Pep

O

R

pyridine

H2O/Me2NPh

Ph-NH-C-HN-CH-C-NH-Pep

O

R

SCF3CO2H

N

S O

RHPh-NH + H3N-Pep

+

a thiazolinone

H2O/H+

NHCH-R

NPh

O

Sphenylthiohydantoin derivative


Specific Cleavage of PeptidesA number of enzymes catalyze the hydrolysis of peptide bonds at specific points in an amino acid sequence. These enzymes are called proteases, peptidases or proteolytic enzymes.

Trypsin is an example. It catalyzes the hydrolysis of polypeptides at the acyl group of arginine or lysine residues.

Chymotrypsin cleaves at the acyl side of the aryl substituted acids phenylalanine, tyrosine and tryptophan.


C-terminal residue

The enzyme carboxypeptidase cleaves the C-terminal amide bond in a peptide. The peptide is incubated with the enzyme until the first amino acid is detected.


Treatment of a peptide with 2,4-dinitrofluorobenzene followed by hydrolysis gives N-dinitrophenylvaline. Leucine is the free acid.

The peptide contains one of each of the following amino acid residues Leu, Ser, Phe, Pro, Tyr, Lys, Gly and Val.

Partial hydrolysis gives four peptides which contain the indicated amino acids:

peptide A:- Leu, Ser, Phe peptide B:- Ser, Pro, Tyr, Lys

peptide C:- Tyr, Lys, Gly peptide D:- Lys, Val, Gly

A peptide!


A peptide has the following amino acid composition: 2 Arg, 2 Ile, Glu, 2 Gly, Leu, Lys, Phe, Pro, Ser, Trp Reaction with Edman’s reagent releases PTH-Leu. Trypsin, a digestive enzyme of intestinal liquids, cleaves polypeptides only at the carboxy (acid) end of arginine (Arg) and Lysine (Lys). Chymotrypsin which is also found in mammalian intestines, cleaves the carboxy end of phenylalanine (Phe), tryptophan (Trp) and tyrosine (Tyr). Hydrolysis by trypsin produces Gly-Arg, Ile-Trp-Phe-Pro-Gly-Arg, Leu-Lys, and Ser-Glu-Ile. Hydrolysis by chymotrypsin produces one peptide with a partial sequence of Leu-Lys-Gly … and another with partial sequence Phe-Pro-Gly-Arg-Ser ...

© E.V. Blackburn, 2012 Amino Acids and Proteins. © E.V. Blackburn, 2012 The hydrolysis of most...

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Transcript of © E.V. Blackburn, 2012 Amino Acids and Proteins. © E.V. Blackburn, 2012 The hydrolysis of most...