bio.lesson document 10

8/6/2019 bio.lesson document 10

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BIO 203 Biochemistry Iby

Seyhun YURDUGL,Ph.D.

Lecture 4The Chemistry of Amino Acids


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Content Outline

Aminoacids

Classifications of Amino Acids

The Isoelectric Point Optical Properties of the Amino Acids

The Peptide Bond

Separation of Amino Acids


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Aminoacids(aa)

The building blocks of proteins or polypeptide

chains All peptides and polypeptides: polymers of

alpha-amino acids.

20 -amino acids relevant to the make-up ofmammalian proteins


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Chemical nature of the amino acids

Several other amino acids : found in the body freeor in combined states

(i.e. not associated with peptides or proteins).

These non-protein associated amino acidsperform specialized functions.


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Structure of amino acids

The -amino acids in peptides and proteins(excluding proline) consist of

a carboxylic acid (-COOH) and an amino (-NH2) functional group

attached to the same tetrahedral carbon atom.

This carbon : -carbon.


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Structure of amino acids

Distinct R-groups, that distinguish one amino

acid from another,

also attached to the -carbon except in the case of glycine where the R-

group: hydrogen

The fourth substitution on the tetrahedral -

carbon of amino acids is hydrogen.


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Aminoacids in action

Several of the amino acids found in proteins:

also serve functions distinct from the formation of

peptides and proteins, e.g. tyrosine

in the formation of thyroid hormones

or glutamate

acting as a neurotransmitter.


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Classifications of

Amino Acids(a.a.)

Each of 20 -amino acids

can be distinguished by the R-group substitution on the-carbon atom.

At present two broad classes of amino acids: basedupon whether the R-group:

is hydrophobic

or hydrophilic.


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Classifications of

Amino Acids

hydrophobic aa tend to repel the aqueousenvironment

and, therefore, reside predominantly in theinterior of proteins.

these amino acids does not ionize norparticipate in the formation of H-bonds.


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Classifications of

Amino Acids

hydrophilic aa: tend to interact with theaqueous environment,

often involved in the formation of H-bonds predominantly found on the exterior surfaces

of :

proteins or in the reactive centers of enzymes.


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Acid-Base properties of the amino

acids

The -COOH and -NH2 groups in amino acids:

capable of ionizing

as are the acidic and basic R-groups of the amino acids

the following ionic equilibrium reactions may bewritten due to ionizability:

R-COOH R-COO- + H+

R-NH3+ R-NH2 + H+


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Acid-Base properties of the amino acids

The equilibrium reactions demonstrate that:

amino acids contain at least two weakly acidic groups.

However, the carboxyl group:

a far stronger acid than the amino group.

At physiological pH (around 7.4) the carboxyl group:unprotonated

and the amino group will be protonated.


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Acid-Base Properties of the Amino Acids

An amino acid with no ionizable R-group is electricallyneutral at a pH:

termed zwitterion. Like typical organic acids, the acidic strength of the

carboxyl, amino and ionizable R-groups in amino

acids:

can be defined by the association constant, Ka

or more commonly the negative logarithm of Ka, the

pKa.


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Acid-Base properties of the amino

acids

net charge:

the algebraic sum of all of the charged groupspresent) of any amino acid, peptide or

protein,

depend upon the pH of the surrounding

aqueous environment.


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As the pH of an amino acid or protein solutionchanges:

so too does the net charge: can be observed during the titration of any amino

acid or protein.

When the net charge of an amino acid or protein =

zero the pH will be equivalent to the isoelectric point: pI.

TheThe isoelectricisoelectric pointpoint

((pIpI))


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Titration curve for alanine


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Functional significance of amino acid R-groups

What are R-groups required for?

In solution

the nature of the amino acid R-groups dictate

structure-function relationships of peptides andproteins:

hydrophobic amino acids generally be encountered inthe interior of proteins:

shielded from direct contact with water.


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Functional significance of amino acid R-groups:

What are R-groups required for?

Conversely, the hydrophilic amino acids: found on the

exterior of proteins as well as in the active centers ofenzymatically active proteins.

Indeed, naturally certain amino acid R-groups:

allow enzyme reactions to occur.


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Examples from different R-groups

The imidazole ring of histidine:

allows it to act as either a proton donor or acceptor atphysiological pH:

frequently found in the reactive center of enzymes.

Equally important:

ability of histidines in hemoglobin to buffer the H+ ionsfrom carbonic acid ionization in red blood cells.

It is this property of hemoglobin that allows it to exchange

O2 and CO2 at the tissues or lungs, respectively.


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Examples from different R-groups

The primary alcohol of serine and threonine as well as thethiol (-SH) of cysteine:

allow these amino acids to act as nucleophiles during

enzymatic catalysis. Additionally, thiol of cysteine:

able to form a disulfide bond with other cysteines:

Cysteine-SH + HS-Cysteine Cysteine-S-S-Cysteine


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Properties of Cystine

This simple disulfide :

identified as cystine.

The formation of disulfide bonds between cysteines presentwithin proteins:

important to the formation of active structural domains in alarge number of proteins.

Disulfide bonding between cysteines in different polypeptidechains of oligomeric proteins:

tend to order the structure of complex proteins, e.g. theinsulin receptor.


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Optical properties of the amino

acids A tetrahedral carbon atom with four(4) distinctconstituents is said to be chiral.

The only one amino acid not exhibiting chirality:

glycine

since its '"R-group" is a hydrogen atom.


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acids Chirality describes: handedness of a molecule that is observable by

the ability of a molecule to rotate the plane of

polarized light either to the right (dextrorotatory)

or to the left (levorotatory).

All of the amino acids in proteins exhibit the

same absolute steric configuration as L-glyceraldehyde.


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acids Therefore, they are all L-alpha-amino acids. D-amino acids :

never found in proteins, although they existin nature.

D-amino acids are often found in polypeptide

antibiotics.


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acids The aromatic R-groups in amino acids: absorb ultraviolet light(U.V.) with an absorbance

maximum in the range of 280 nm.

The ability of proteins to absorb U.V. light: predominantly due to the presence of thetryptophan

which strongly absorbs U.V. light.


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The peptide bond:

Peptide bond formation:

a condensation reaction

leading to the polymerization of amino acids intopeptides and proteins.


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The peptide bond:

Peptides are small consisting of few amino acids.

e.g. A number of hormones and neurotransmitters,

several antibiotics and antitumor agents.

Proteins: polypeptides of greatly divergent length.


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The peptide bond:

The simplest peptide, a dipeptide:

contains a single peptide bond

formed by the condensation of the carboxyl group of

one amino acid with the amino group of the second with the

concomitant elimination of water.


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The peptide bond

The presence of the carbonyl group in a peptide bond:

allows electron resonance stabilization to occur:

such that the peptide bond exhibits rigidity not unlike

the typical -C=C- double bond. The peptide bond is, therefore, said to have partial

double-bond character.


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Structure of peptide bond:

Resonance stabilization forms of peptide bond


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Solubility of proteins

Proteins show a variation in solubility:

depending on their solution ionic

environments. This property:

characterized in:

salting-in, salting-out, and dialysis of proteins.


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Salting-in:

The effects of salts such as sodium chloride;

on increasing the solubility of proteins: often referred to as salting-in.


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Salting-in

When low concentrations of salt:

added to a protein solution,

the solubility increases. This could be explained by the following:


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Salting-in

Salt molecules stabilize protein molecules by:

decreasing the electrostatic energy:

between the protein molecules; which increase the solubility of proteins.


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Ionic strength

measures the concentration of charges insolution.

As the ionic strength of a solution increases, the activity coefficient of an ion decreases.


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Ionic strength

Formula:

/2 = Mi Zi2

Mi= the molarity of the ion

Zi= the net charge of the ion(regardless of itssign)

= the sum

= Gamma letter in Greek


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Ionic strength

The relationship between the ionic strengthand the molarity of a solution of ionizable salt:

depends on the number of ions produced andtheir net charge.


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Salt type Salt example Ionic strength

1:1 KCl, NaBr M

2:1 CaCl2, Na2HPO4 3XM

2:2 MgSO4 4XM 3:1 FeCl3 6XM

2:3 Fe2(SO4)3 15XM


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Ionic strength

Type:

refers to the net charge on the ions.

Only the net charge on an ion: used in calculating the ionic strength.


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Salting-out:

When the ionic strength of a protein solution:

increased by adding salt,

the solubility decreases, and protein precipitates.

This could be explained by the following:


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Salting-out:

The salt molecules compete with the proteinmolecules;

in binding with water.

Some salts, such as high concentrations ofammonium sulfate,

have general effects on solvent structure that

lead to: decreased protein solubility and salting-out.


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Salting-out:

In this case, the protein molecules tend to associate with each

other; because protein-protein interactions become

energetically more favorable than protein-solventinteraction.

Proteins have characteristic salting-out points, and these:

used in protein separations in crude extracts.


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Salting-out:

The most effective region of salting-out:

at the isoelectric point of the protein:

because all proteins exhibit minimumsolubility in solutions of constant ionicstrength;

at their isoelectric points.


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Salting-out:

The salt commonly used:

ammonium sulfate because of:

its large solubility in water. its relative freedom from temperature effects.

it has no harmful effects on most of the

proteins.


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Various techniques in separation ofamino acids

A) Ion-exchange chromatography.

Depends on electrical charge

two main mechanisms for effecting separationin ion-exchange chromatography.

One is titration,

The other: increasing the pH.


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Ion exchange chromatography apparatus


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Separation of amino acids

As pH increases,

the amino acids go from a positive to a neutralcharge state,

and so released from the fixed anionic sites in

the resin (stationary phase)


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One other mechanism:

competitive ion exchange.

The positive amino acids show a higher affinity for

the ion-exchange sites than do the eluting cations(H+, Li+ or Na+)

and so are bound strongly to the anionic sites.

eluted by the continuous flow of cation;

or by increasing cation concentration developed bygradient formation.


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Separation of Amino Acids

Amino acid analyzers, such as the BeckmanSystem 6300TM

based on step gradients-- isocratic procedures

that step through a set of 3-6 solutions ofvarying pH and cation concentration.

Both titration and elution adjustments are

made with each eluant step.


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The modern liquid chromatographic-highperformance liquid chromatography (HPLC)techniques

Modern enhancement of ion exchangechromatography:

use three solutions and continuous gradients.

HPLC is; to a lesser extent; also called as: highpressure liquid chromatography.


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HPLC (High Performance LiquidChromatography)


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An amino acid analyzer: a special instrumentfor the separation process.

Gradient capability allows for easier methodsdevelopment and results in flatter baselines--especially important at high sensitivity.

As with dedicated analyzers, the eluants are

either sodium- or lithium-based.


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Amino acid analyzer


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LITERATURE CITED

Devlin,T.M. Textbook of Biochemistry with Clinical

Correlations,Fifth Edition,Wiley-Liss Publications,New

York, USA, 2002.

Lehninger, A. Principles of Biochemistry, Second edition,

Worth Publishers Co., New York, USA, 1993.

Matthews, C.K. and van Holde, K.E., Biochemistry,

Second edition, Benjamin / Cummings Publishing

Company Inc., San Francisco, 1996.

Segel, I.H., Biochemical Calculations, Wiley Publications,New York, USA, 1976.

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bio.lesson document 10