Ionization and Ph of Amino Acid

Assignment on "Ionization and PH of Amino Acid"

1. 2
ASSIGNMENT ON “IONIZATION AND PH
OF AMINO ACID”





Amino acids are biologically important
organic compounds composed of amine (-NH2)
and carboxylic acid (-COOH) functional groups,
along with a side-chain specific to each amino
acid. The key elements of an amino acid are
carbon, hydrogen, oxygen, and nitrogen, though
other elements are found in the side-chains of
certain amino acids. About 500 amino acids Fig: Amino Acid
are known and can be classified in many ways.
They can be classified according to the core structural functional groups'
locations as alpha- (α-), beta- (β-), gamma- (γ-) or delta- (δ-) amino acids; other
categories relate to polarity, pH level, and side-chain group type (aliphatic,
acyclic, aromatic, containing hydroxyl or sulfur, etc.). In the form of proteins,
amino acids comprise the second-largest component (water is the largest) of
human muscles, cells and other tissues. Outside proteins, amino acids perform
critical roles in processes such as neurotransmitter transport and biosynthesis.
Amino acids having both the amine and the carboxylic acid groups attached to
the first (alpha-) carbon atom have particular importance in biochemistry. They
are known as 2-, alpha-, or α-amino acids (generic formula H2NCHRCOOH in
most cases

where R is an organic substituent known as a "side-chain"); often the
term "amino acid" is used to refer specifically to these. They include the 23

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proteinogenic ("protein-building") amino acids, which combine into peptide
chains ("polypeptides") to form the building-blocks of a vast array of proteins.
These are all L-stereoisomers ("left-handed" isomers), although a few D-amino
acids ("right-handed") occur in bacterial envelopes and some antibiotics.
Twenty of the proteinogenic amino acids are encoded directly by triplet codons
in the genetic code and are known as "standard" amino acids. The other two
("non-standard" or "non-canonical") are pyrrolysine (found in methanogenic
organisms and other eukaryotes) and selenocysteine (present in many
noneukaryotes as well as most eukaryotes). For example, 25 human proteins
include selenocysteine (Sec) in their primary structure, and the structurally
characterized enzymes (selenoenzymes) employ Sec as the catalytic moiety in
their active sites. Pyrrolysine and selenocysteine are encoded via variant
codons; for example, selenocysteine is encoded by stop codon and SECIS
element. Codon–tRNA combinations not found in nature can also be used to
"expand" the genetic code and create novel proteins known as alloproteins
incorporating non-proteinogenic amino acids.
Many important proteinogenic and non-proteinogenic amino acids also
play critical non-protein roles within the body. For example, in the human brain,
glutamate (standard glutamic acid) and gamma-amino-butyric acid ("GABA",
non-standard gamma-amino acid) are, respectively, the main excitatory and
inhibitory neurotransmitters; hydroxyproline (a major component of the
connective tissue collagen) is synthesised from proline; the standard amino acid
glycine is used to synthesise porphyrins used in red blood cells; and the non-
standard carnitine is used in lipid transport. Nine proteinogenic amino acids are
called "essential" for humans because they cannot be created from other
compounds by the human body and, so, must be taken in as food. Others may be
conditionally essential for certain ages or medical conditions. Essential amino
acids may also differ between species. Because of their biological significance,
amino acids are important in nutrition and are commonly used in nutritional

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supplements, fertilizers, and food technology. Industrial uses include the
production of drugs, biodegradable plastics, and chiral catalysts.
The first few amino acids were discovered in the early 19th century. In
1806, French chemists Louis-Nicolas Vauquelin and Pierre Jean Robiquet
isolated a compound in asparagus that was subsequently named asparagines, the
first amino acid to be discovered. Cystine was discovered in 1810, although its
monomer, cysteine, remained undiscovered until 1884. Glycine and leucine
were discovered in 1820. Usage of the term amino acid in the English language
is from 1898. Proteins were found to yield amino acids after enzymatic
digestion or acid hydrolysis. In 1902, Emil Fischer and Franz Hofmeister
proposed that proteins are the result of the formation of bonds between the
amino group of one amino acid with the carboxyl group of another, in a linear
structure that Fischer termed peptide.


Classifications:
Experts classify amino acids based on lots of different features. One of
them is whether or not people can acquire them through the diet. According to
this factor, scientists recognize 3 types: the nonessential, essential, and
conditionally essential amino acids. However, the classification as essential or
nonessential doesn't actually reflect their importance, as all twenty of them are
necessary for human health. Those 8 called essential (or indispensable) can't be
produced by the body and therefore should be supplied by food: Leucine,
Isoleucine, Lysine, Threonine, Methionine, Phenylalanine, Valine, and
Tryptophan. One more amino acid, Histidine, can be considered semi-essential,
as the human body doesn't always need dietary sources of it. Meanwhile,
conditionally essential amino acids aren't usually required in the human diet, but
are able to become essential under some circumstances. Finally, nonessential
ones are produced by the human body either out of the essential ones or from

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normal proteins breakdown. These include Asparagine, Alanine, Arginine,
Aspartic acid, Cysteine, Glutamic acid, Glutamine, Praline, Glycine, Tyrosine,
and Serine.
One more classification depends on the side chain structure, and experts
recognize 5 types in this classification:

I. Containing sulfur (Cysteine and Methionine)
II. Neutral (Asparagine, Serine, Threonine, and Glutamine)
III. Acidic (Glutamic acid and Aspartic acid) and basic (Arginine and
Lysine)
IV. Alphatic (these include Leucine, Isoleucine, Glycine, Valine, and
Alanine)
V. Aromatic (these include Phenylalanine, Tryptophan, and Tyrosine)


 NEUTRAL SIDE CHAINS:
These amino acids are characterised
by two pKa : pKa1 and pKa2 for the
carboxylic acid and the amine respectively. Fig.: 3D structure of Asparagine
The isoelectronic point will be halfway between,
or the average of, these two pK as, i.e. pI = 1/2 (pKa1 + pKa2). This is most
readily appreciated when you realise that at very acidic pH (below pKa1) the
amino acid will have an overall +ve charge and at very basic pH (above pKa2 )
the amino acid will have an overall -ve charge. For the simplest amino acid,
glycine, pKa1= 2.34 and pKa2 = 9.6, pI = 5.97.

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The other two cases introduce other ionisable groups in the side chain "R"
described by a third acid dissociation constant, pKa3
 ACIDIC SIDE CHAINS
The pI will be at a lower pH because the acidic
side chain introduces an "extra" negative
charge. So the neutral form exists under more
acidic conditions when the extra -ve has been
neutralised. For example, for aspartic acid Fig.: 3D structure of Glutamic acid
shown below, the neutral form is dominant between pH 1.88 and 3.65, pI is
halfway between these two values, i.e. pI = 1/2 (pKa1 + pKa3), so pI = 2.77.







 BASIC SIDE CHAINS
The pI will be at a higher pH because the
basic side chain introduces an "extra"
positive charge. So the neutral form
exists under more basic conditions Fig.: 3D structure of Histidine
when the extra +ve has been neutralized.
For example, for histidine, which was discussed on the previous page, the
neutral form is dominant between pH 6.00 and 9.17, pI is halfway between
these two values, i.e. pI = 1/2 (pKa2 + pKa3), so pI = 7.59

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Different Amino Acid with Symbol, Structure ETC.:

Amino
Acid

Symbol

Structure

pK1
(COOH)

pK2
(NH2)

pK
R Group
Amino Acids with Aliphatic R-Groups
Glycine Gly – G

2.4 9.8
Alanine Ala – A

2.4 9.9
Valine Val – V

2.2 9.7
Leucine Leu – L

2.3 9.7
Isoleucine Ile – I

2.3 9.8
Non-Aromatic Amino Acids with Hydroxyl R-Groups
Serine Ser – S

2.2 9.2 ≈13
Threonine Thr – T

2.1 9.1 ≈13
Amino Acids with Sulfur-Containing R-Groups

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Cysteine Cys – C

1.9 10.8 8.3
Methionine Met –M

2.1 9.3
Acidic Amino Acids and their Amides
Aspartic
Acid
Asp – D

2.0 9.9 3.9
Asparagine Asn – N

2.1 8.8
Glutamic
Acid
Glu – E

2.1 9.5 4.1
Glutamine Gln – Q

2.2 9.1

Basic Amino Acids
Arginine Arg – R

1.8 9.0 12.5
Lysine Lys – K

2.2 9.2 10.8
Histidine His – H

1.8 9.2 6.0
Amino Acids with Aromatic Rings
Phenylala
nine
Phe – F

2.2 9.2

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Ionization of Amino acids:

Ionization is the process by which an atom or a molecule acquires a
negative or positive charge by gaining or losing electrons. Ionization, often,
results from the interaction of an atom or a molecule with an ionizing particle,
including charged particles with sufficient energies and energetic photons. A
rare case of ionization in the absence of an external particle is the internal
conversion process, through which an excited nucleolus transfers its energy to
one of the inner-shell electrons and ejects it with high kinetic energy. The
ionization process is of particular interest in fundamental science. It is also
encountered in many fields of practical interest, ranging from mass
spectroscopy to radiotherapy for eliminating cancer cells. Obviously, all aspects
of the ionization process cannot be addressed in a single encyclopedic article.
Here, ionization is described by invoking the simple case of an atom in strong
laser field. The case is amenable to analytic treatment and represents the main
Tyrosine Tyr – Y

2.2 9.1 10.1
Tryptophan Trp – W

2.4 9.4

Imino Acids
Proline Pro – P

2.0 10.6

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characteristics of the process. The α-carboxyl and α-amino groups on amino
acids act as acid–base groups, donating or accepting a proton as the pH is
altered. Both groups are fully protonated, at low p
H
, but as the p
H
is increased
first the carboxyl group and then the amino group loses a hydrogen ion. The pK
is in the range 1.8–2.9 for the α-amino group for the α-carboxyl group and 8.8–
10.8 for the standard 20 amino acids. Those amino acids with an ionizable side-
chain are having an additional acid–base group with a distinctive pK. The
twenty standard amino acids contain two acid–base groups: the α- carboxyl and
α-amino groups associated to the Cα atom. Those amino acids with an ionizable
side-chain (Glu, Asp, Lys, Arg His, Tyr, Cys) have an additional acid–base
group. The titration curve of Gly is shown in given figure. At low p
H
(for
example high hydrogen ion concentration) both the amino group and the
carboxyl group are completely protonated so that in the cationic form H3N +
CH2 COOH the amino acid is as in solution the amino acid is titrated with
increasing amounts of a strong base (for example NaOH), it loses 2 protons
As a result from the carboxyl group which has the lower pK value (pK = 2.3)
and then from the amino group which has the higher pK value (pK = 9.6). The
pH at which Gly has no net charge is termed its isoelectric point, pI. The α-
carboxyl groups of all the twenty standard amino acids contain pK values in
the range 1.8–2.9, whereas their α-amino groups
contain pK values in the range 8.8–10.8 .The side-chains of the acidic
amino acids Glu and Asp contain pK values of 4.1 and 3.9,
respectively, whereas those of the basic amino acids Arg and Lys, contain
pK values of 12.5 and 10.8, respectively. The side-chain of His, Only with a pK
value of 6.0, is ionized within the physiological pH range (p
H
6–8). It should be
kept in mind that when the amino acids are attached together in proteins, the
side-chain groups and the terminal α-amino and α-carboxyl groups are free to
ionize only.

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Ionization of glycine. (a) Titration curve of glycine, (b) dissociation of glycine.
Numbers in bold in parentheses in (a) correspond to the structures in (b).

Table: pK values and molecular weights of the 20 standard amino acid

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Isoelectronic point (pI):
The isoelectronic point or isoionic point is the p
H
at which the amino
acid does not migrate in an electric field.
This means it is the pH at which the amino acid is neutral, i.e. the
zwitterion form is dominant.
A table of pKa and pI values can be found on the next page.
The pI is given by the average of the pK as that involve the zwitterion,
i.e. that give the boundaries to its existence.
The Isoelectric Point or pI is a P
H
solution in which an amino acid are
present as zwitterion and net charge is zero. Each amino acid has a fixed and
constant Isoelectric Point. E.g. For alanine, pI is 6.01
The net charge on an acid depends on the P
H
of the solution in which it
is dissolved.
P
H
: 5 to 6 isoelectric point (pI)

Positive charge = Negative charge
No net charge- Zwitterion

P
H
: about 2 to 3 -COO
-
acts as a base and accepts an H
+


+ H3O
+
+ H2O

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P
H
: about 7.6 to 10.8 -NH3
+
acts as a acid and loses an H
+



+ OH
-
+ H2O



Each Amino Acid has a fixed and constant pI and that’s given below:
Nonpolar & Polar side chains pI
Alanine 6.01
Asparagine 5.41
Cysteine 5.07

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Zwitterions:

When an amino acid molecule contains a positive and a negative charge
then it is termed as zwitterion. They have no net charge. In solution, amino acid
becomes charged. This is because of having an acidic group (-COOH) and a
basic group (-NH2) in the same molecule. Transfer of proton takes place in a
Glutamine 5.65
Glycine 5.97
Isoleucine 6.02
Leucine 6.02
Methionine 5.74
Phenylalanine 5.48
Proline 6.48
Serine 5.68
Threonine 5.87
Tyrosine 5.66
Tryptophan 5.89
Valine 5.97
Acidic Side Chains pI
Aspartic Acid 2.77
Glutamic Acid 3.22
Basic Side Chains pI
Arginine 10.76
Histidine 7.59
Lysine 9.74

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kind of internal acid base reaction. The production of this reaction is called
zwitterion.







The amine and carboxylic acid functional groups found in amino acids allow
them to have amphiprotic properties. Carboxylic acid groups (−CO2H) can be
deprotonated to become negative carboxylates (−CO2
−
), and α-amino groups
(NH2−) can be protonated to become positive α-ammonium groups (
+
NH3−). At
pH values greater than the p
Ka
of the carboxylic acid group (mean for the 20
common amino acids is about 2.2, see the table of amino acid structures above),
the negative carboxylate ion predominates. At p
H
values lower than the pKa of
the α-ammonium group (mean for the 20 common α-amino acids is about 9.4),
the nitrogen is predominantly protonated as a positively charged α-ammonium
group. Thus, at p
H
between 2.2 and 9.4, the predominant form adopted by α-
amino acids contains a negative carboxylate and a positive α-ammonium group,
as shown in structure (2) on the right, so has net zero charge. This molecular
state is known as a zwitterion, from the German Zwitter meaning hermaphrodite
or hybrid. Below pH 2.2, the predominant form will have a neutral carboxylic
acid group and a positive α-ammonium ion (net charge +1), and above p
H
9.4, a
negative carboxylate and neutral α-amino group (net charge −1). The fully
neutral form (structure (1) on the right) is a very minor species in aqueous
solution throughout the p
H
range (less than 1 part in 10
7
). Amino acids exist as
zwitterions also in the solid phase, and crystallize with salt-like properties
unlike typical organic acids or amines. At p
H
values between the two pKa
values, the zwitterion predominates, but coexists in dynamic equilibrium with

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small amounts of net negative and net positive ions. At the exact midpoint
between the two pKa values, the trace amount of net negative and trace of net
positive ions exactly balance, so that average net charge of all forms present is
zero. This pH is known as the isoelectric point pI, so pI = ½(pKa1 + pKa2). The
individual amino acids all have slightly different pKa values, so have different
isoelectric points. For amino acids with charged side-chains, the pKa of the
side-chain is involved. Thus for Asp, Glu with negative side-chains, pI =
½(pKa1 + pKaR), where pKaR is the side-chain pKa. Cysteine also has
potentially negative side-chain with pKaR = 8.14, so pI should be calculated as
for Asp and Glu, even though the side-chain is not significantly charged at
neutral pH. For His, Lys, and Arg with positive side-chains, pI = ½(pKaR +
pKa2). Amino acids have zero mobility in electrophoresis at their isoelectric
point, although this behaviour is more usually exploited for peptides and
proteins than single amino acids. Zwitterions have minimum solubility at their
isolectric point and some amino acids (in particular, with non-polar side-chains)
can be isolated by precipitation from water by adjusting the pH to the required
isoelectric point.






Effect of pH on Structure of Amino Acids
Amino acids are characterized by the presence of both acidic (carboxylic
acid) and basic (amine) functional groups. These groups undergo the same type
of equilibrium reactions that all weak acids and bases undergo, and the relative
amount of each form can be altered by adjusting the p
H
of the solution. For
example,

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RCOOH <----> RCOO
-1
+ H
+1

R-NH3
+1
<----> R-NH2 + H
+1

Both of the reactions shown above can be shifted to the left by increasing the
concentration of acid (i.e. - lowering the solution p
H
) as predicted by Le
Chatlier's principle. Conversely, increasing the solution p
H
to give a more basic
(less acidic) solution will shift the equilibrium to the right. For this reason, the
organic compounds shown on the left in each of the above reactions are
sometimes referred to as the 'acid forms' while the organic compounds on the
right are called the 'base forms'. This convention is consistent, since the 'base
forms' react with acid to produce the 'acid forms'.
The relative concentrations of each form can be calculated using the Henderson-
Hasselbalch equation:

Since the log10 of 1.0 is equal to 0.0, when the solution P
H
is set equal to the pKa
of the functional group, the relative amounts of acid and base forms should be
equal. If the P
H
is lowered (more acidic), the relative amount of the acid form
(HA) will increase with a corresponding decrease in the concentration of the
base form (A
-
).If the pH is increased (more basic), the opposite trend should be
observed. A plot of concentration vs. P
H
can be produced that indicates the
relative amounts of each form. If the concentrations of both forms are included
on this graph, the curves will cross (50% of each) when the P
H
is equal to the
pKa. Shown below is a plot showing the relative amounts of each form of
glycine (the simplest amino acid) as a function of P
H
. Since amino acids contain
a minimum of two functional groups, two sets of curves are required. Above the
plot, the predominant structure for each pH region is shown.

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