قسم الكيمياء الحيوية

سلامه.د على ى .دولت و الحيوية استاذ الكيمياء٢٠١٥-٢٠١٤

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المحاضرة الثانية

Content :• Properties of amino acids..

S i d A l i f A i A id• Separation and Analysis of Amino Acids

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Physical Properties of Amino acidsy1.Solubility:

All amino acids are soluble in water due to the presenceAll amino acids are soluble in water due to the presence of amino, carboxyl and other hydrophilic groups.

2.Optical activity:Due to the presence of asymmetric carbon atoms allDue to the presence of asymmetric carbon atoms all amino acids expect for glycine are optically active.

4• All amino acids found in proteins are of the L-configuration.

3. Melting points : Amino acids generally melt at higher temparatures often above 200ºChigher temparatures, often above 200 C.

4.Taste :Amino acids may be sweet( Gly, Ala, Val ), tasteless ( Leu ) or bitter ( Arg, Ile ).

– Monosodium glutamate is a salt of glutamic acid. • It is employed as a flavoring agent in food

industry to increase taste and flavorindustry to increase taste and flavor

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55.ACIDIC .ACIDIC AND BASIC PROPERTIES AND BASIC PROPERTIES OF AMINO ACIDSOF AMINO ACIDS

• The general properties of amino acids are:

OF AMINO ACIDS OF AMINO ACIDS The general properties of amino acids are:– They can act as acids (due to the carboxylic acid

group) and bases (due to the amine group), and thus they are excellent buffersthey are excellent buffers

– They migrate when an electric field is applied

• Amino acids exist as highly polar ions that are called zwitterions (“double-ions”)

Zwitterions are ions that have both a negative and a– Zwitterions are ions that have both a negative and a positive charge within the same structure

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Isoelectric Point• Each amino acid has an isoelectric point, (pI)

numerically equal to the pH at which the zwitteriont ti i t iconcentration is at a maximum.

• The amino acid has no NET charge at its pI; it has one positive and one negative chargepositive and one negative charge.

• At a pH less than the value of the isoelectric point, the amino acid is protonated and has a POSITIVE charge; atamino acid is protonated and has a POSITIVE charge; at a pH greater than the pI the amino acid is deprotonated and has a NEGATIVE charge.g

C

H3N H

O OC

H3N H

O OHC

H2N H

O OH OH

C ti N t l Anion

3

R3

R2

R

@ pH = pI@ pH < pI @ pH > pI

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Cation Neutral Anion

(zwitterion form)

If the zwitterion is treated with acid, that is, a solution containing H+ , the H+ will add to the COO- to form COOH.

Si il l t ti th itt i ith b ill• Similarly, treating the zwitterion with base will result in the loss of the removable proton attached to the NH3

+ group to form NH2 .attached to the NH3 group to form NH2 .

• The pH at which amino acids have no net pcharge is called the Isoelectric Point or the pI.

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Amino Acids Above and Below Their Isoelectric Points

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Henderson-Hasselbalch• We have calculated the ratio of acid to conjugate

base for an -carboxyl group and an -amino groupat pH 7.0

• We can do this for any weak acid and its conjugate base at any pH using the Henderson-Hasselbalch equation

pH= pKa+ log [unprotonated form (conjugate base)][protonated form (weak acid) ]

H-H equation can be used to calculate the average charge on an ionizable group at any pH

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g g g p y p

L t’ i th b h i f W k A id( HA) i l tiLet’s examine the behavior of a Weak Acid( HA), in aqueous solution.

R l h H K d [A ]/[HA] If kRelates three terms: pH, pKa, and [A‐]/[HA]. If you know two of these values, you can determine the third.

pH = pKa + log([A‐]/[HA])When [A‐] = [HA]:

pH = pKa + log(1)pH = pKa + 0p p a

pH = pKa pka is the pH at which a functional group existsfunctional group exists 50% in its protonated form (HA) and 50% in its d t t d f (A )

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deprotonated form (A‐).

pI Depends on Side Chain

The 15 amino acids thiol, hydroxyl groups or pure hydrocarbon side chains have pI = 5.0 to 6.5 (average of the pKa’s)

G Glu and Asp have acidic side chains and a lower PI His, Arg, Lys have basic side chains and higher pI

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22.Write .Write out structures for sequential deprotonation out structures for sequential deprotonation d l K ld l K l th ilib ith ilib iand place pKa values over and place pKa values over the equilibrium the equilibrium arrows.arrows.

Fully protonated 1st proton removed 2nd proton removedFully protonated 1st proton removed 2nd proton removedNet charge = +1 Net charge = 0 Net charge = -1

So, from looking at the net charges, at different pH’s, amino 1313

So, o oo g a e e c a ges, a d e e p s, a oacids can have different charges!

Very important for protein structure!!

Q:WriteQ:Write TheThe charges of the lysine at different pHcharges of the lysine at different pHQ:Write Q:Write The The charges of the lysine at different pHcharges of the lysine at different pH

At pH=At pH=11 the lysine possesses two positive atthe lysine possesses two positive at At pH=At pH=1 1 the lysine possesses two positive, at the lysine possesses two positive, at pH=pH=55..6 6 two positive and one negative, at pH=two positive and one negative, at pH=99..7 7 one positive and one negative and atone positive and one negative and at pH=pH=1111 oneoneone positive and one negative and at one positive and one negative and at pH=pH=11 11 one one negative negative

The lysine has an amino group on its side chainThe lysine has an amino group on its side chain The lysine has an amino group on its side chain, The lysine has an amino group on its side chain, its isoelectric point is at pH=its isoelectric point is at pH=99..77.. 1414

Aspartate Aspartate (D, Asp): (D, Asp): Asp Asp has has 3 3 titratable protonstitratable protons

Amino acids can be separated on the basis of their charges at a certain pH

1. pKa’s for the three groups (look at Table23.2)2. Draw the structures from fully protonated to fully deprotonatedN t th t ll i id t i t l t i ll t l t HNote that all amino acids are at one point, electrically neutral at some pH value.

This pH = isoelectric point (pI)

1515pKas: 2, 3.9, 10 (from Table 3.2)pI = (2+3.9)/2 = 2.95

A buffer is a solution that resists change in pH following the addition of an acid or base A bufferA buffer is a solution that resists change in pH following the addition of an acid or base. A buffer can be created by mixing a weak acid (HA) with its conjugate base (A–).

- If an acid such as HCl is then added to such a solution, A– can neutralize it, in the process being converted to HAbeing converted to HA.

- If a base is added, HA can neutralize it, in the process being converted to A–. • a buffer as a solution that consists of a mixture of a weak acid and it conjugate base

Amino acids have both a carboxylic acid group (acid) and an amine group (base). Those two f nctional gro ps can act as "pse do" b ffer sites If an acid is added the amine can pick pfunctional groups can act as "pseudo" buffer sites. If an acid is added, the amine can pick up the extra protons, and if a base is added, the carboxylic acid group can sort of neutralize it. But amino acids, by themselves, do not fit the definition of a buffer.

if b i dd d If an acid is addedif a base is added

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• Q. Draw the fully protonated structure of Alanine?

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Learning Check AA2g

CH3 CH3

+H3N–CH–COOH H2N–CH2–COO–

(1) (2)Select from the above structuresA. Alanine in base.B Al i i idB. Alanine in acid.

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Solution AA2

CH3 CH3

+H3N–CH–COOH H2N–CH2–COO–

(1) (2)Select from the above structuresA. (2) Alanine in base.B (1) Al i i idB. (1) Alanine in acid.

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Learning CheckLearning Check

• Would the following ions of serine exist at a pH above, below, or at pI?

O O O

H3N CH C

CH2

O H3N CH C

CH2

OH H2N CH C

CH2

O

CH2

OH

CH2

OH

CH2

OH

The anser.PH=PI PH < PI PH >PI

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PH=PI PH < PI PH >PI

6.Spectroscopic Properties All amino acids absorb in

the infrared (bond )vibrations).

Only Phe, Tyr, and Trpabsorb in the UVabsorb in the UV (electronic transitions between energy levels).gy )

Absorbance at 280 nm is a good method for determining protein concentration.

• .The UV spectra of the aromatic amino acids at pH 6. Beer’s Law: A l

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A = εcl

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Absorption of light by molecules

• Spectrophotometer

•Wave length of light…. Ultrviolet 200-350nmWave length of light…. Ultrviolet 200 350nm

Visible 400-700

Infra red 700-

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ChemicalChemical reactionsreactionsDue to carboxyl group:1 i id f lt (COON ) ith b & t1. amino acids form salts (COONa) with bases & ester

(-COOR’ ) with alcohol.2 D b l ti f di i2. Decarboxylation: form corresponding amine.

1. Histidine histamine + CO2

2. Tyrosine tyramine + CO2

3. Tryptophan tryptamine + CO2

4. Lysine cadaverine + CO2

5. Glutamic acid GABA + CO2

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AMINO ACIDS The building blocks of proteins

AMINO ACIDSg p

Released from proteins by hydrolysis

(e g with 6M HCl 110ºC 24 h)(e.g. with 6M HCl, 110ºC, 24 h)

Separated by chromatography or electrophoresis Separated by chromatography or electrophoresis

Detected by reaction with ninhydrin (purple colour)

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Separation and Analysis using pI valuesSeparation and Analysis using pI values• Differences in isoelectric points (and therefore charges) are used to

separate mixtures of amino acids by two common methods:p y

- Ion exchange chromatography :Ion exchange resins have charged groups covalently attached to the stationary phase (adsorbent, matrix), either positive or negative. Obviously, if ionizable groups are weak acids or bases, the pH of the buffer determines the charge state of the matrix. g

- Electrophoresis

OH H O H O

H3 C CO

OH

CH2CO2

N + H3N

CH3

H O

COC + H3

CH2CH2CH2CH2NH3

H O

COCNMixture of:buffered at pH 6.0

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A (pI=6.0)D (pI=2.8) K (pI= 9.7)

aspartic acid alanine lysine

Ion Exchange ChromatographyIon Exchange ChromatographyOH H O H O

H3 C CO

CH2CO2

N

A (pI=6.0)

+ H3N

CH3

COC

D (pI=2.8)

+ H3

CH2CH2CH2CH2NH3

COCN

K (pI= 9.7)

Mixture of:buffered at pH 6.0

SO3 K (strongly retained)

SO3A

D (unretained)

(slightly retained, &sulfonated

polystyrene

SO3

D- elutes first followed by A; K+ elutesD- elutes first, followed by A; K+ elutes last, and on lafter pH of buffer is increased and K+ is deprotonated

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increased and K+ is deprotonated.

• But there is a problem in detecting amino acids; they l l d t f th h littlare colorless, and most of them have very little

absorption in the UV region (they have no conjugation, except in the four aromatic amino acids)except in the four aromatic amino acids)

• To overcome this difficulty, amino acids are converted (after separation by ion exchange chromatography) to(after separation by ion exchange chromatography) to a derivative using ninhydrin.

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Separations of Amino Acids

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- Q. Mixture of Val (pI = 6) ; asp ( pI = 2.8,) lys (pI = 9.7) in a buffer at pH = 6. What is the form of each?

CNCH

O

O_+ H3 C

N

CHO

O-H3+

C

N

CHCH2H2C

N OH3+ H3

+

CHCH3H3C

val

CH2C-O

O

asp lysCH2H2C O-

Consider THREE amino acids mixed together in a buffer at pH 6.0

Ninhydrin is used to detect the individual amino acids30

Separation of Amino Acids by Paper Ch t hChromatography

A mixture of amino acids can also be separated on the basis of polarity

Start Chromatographyg p y

SolventSolvent Front

Origin1 2 3 4x

5 6 7 8 932

1 2 3 4 5 6 7 8 9-Identify unknown amino acids using biochemical tests and Rf values

Ch t hChromatography

SolventSolvent Front

x

Origin1 2 3 4x

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1 2 3 4

QuestionQ1.At pH 7, which of the following amino acids have a net positive charge, which have a net negative charge, and which are neutral?Lysine

Phenylalanine

Leucine

2- Explain How:a molecule's charge (amino acids) changes with the pH of its surroundings.

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