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Table of pKa and pI values

The pKa values and the isoelectronic point, pI, are given below for the 20 amino acids.
pKa1= -carboxyl group, pKa2 =-ammonium ion, and pKa3 = side chain
group.
Amino acid
Glycine
Alanine
Valine
Leucine
Isoleucine
Methionine
Proline
Phenylalanine
Tryptophan
Asparagine
Glutamine
Serine
Threonine
Tyrosine
Cysteine
Aspartic acid
Glutamic acid
Lysine
Arginine
Histidine

pKa1
2.34
2.34
2.32
2.36
2.36
2.28
1.99
1.83
2.83
2.02
2.17
2.21
2.09
2.20
1.96
1.88
2.19
2.18
2.17
1.82

pKa2
9.60
9.69
9.62
9.60
9.60
9.21
10.60
9.13
9.39
8.80
9.13
9.15
9.10
9.11
8.18
9.60
9.67
8.95
9.04
9.17

pKa3
------------------------------3.65
4.25
10.53
12.48
6.00

pI
5.97
6.00
5.96
5.98
6.02
5.74
6.30
5.48
5.89
5.41
5.65
5.68
5.60
5.66
5.07
2.77
3.22
9.74
10.76
7.59

Amino Acids with Acidity Values


The structure to the right is a generic amino acid. All of the common amino acids
except glycine are chiral (have a handedness). The form shown (the S-amino acid, or
L-amino acid) is the most common form found in plants and animals. The mirror
image of each amino acid can be found in nature, although they are less common.

The third through fifth columns give pKa values of groups:


-NH3 refers to the protonated -amino group, the CO2H refers to
the carboxylic acid group on the -carbon, and the side chain is
just that. In all cases, these refer to the free amino acid, and not
the amino acid incorporated into a polypeptide or protein chain.
Usually, we assume that not much happens to the pK a of the side chain on
incorporation, as a first guess. While that is not strictly true, there are so many other
environmental effects on the values that this value works well as a starting point, until
the value can be measured independently.

Amino Acid

-NH3+

-CO2H Side chain

pI

Glycine, Gly

-H

9.78

2.35

5.97

Alanine, Ala

-CH3

9.87

2.35

6.02

Valine, Val

-CH(CH3)2

9.74

2.29

5.97

Leucine, Leu

CH2CH(CH3)2

9.74

2.33

5.98

Isoleucine, Ile

CH(CH3)CH2CH3 9.76

2.32

6.02

Phenylalanine, Phe

9.31

2.20

5.48

Tryptophan, Trp

9.41

2.46

5.88

Tyrosine, Tyr

9.21

2.20

10.46

5.65

Histidine, His

9.33

1.80

6.04*

7.58

Serine, Ser

CH2OH

9.21

2.19

5.68

Threonine, Thr

CH(CH3)-OH

9.10

2.09

6.53

Methionine, Met

CH2CH2SCH3

9.28

2.13

5.75

Cysteine, Cys

CH2SH

10.70

1.92

8.37

5.14

Aspartic Acid, Asp

CH2CO2H

9.90

1.99

3.90

2.87

Glutamic Acid, Glu

CH2CH2CO2H

9.47

2.10

4.07

3.22

Asparagine, Asn

CH2CONH2

8.72

2.14

5.41

Glutamine, Gln

CH2CH2CONH2

9.13

2.17

5.65

Lysine, Lys

(CH2)4NH2

9.06

2.16

10.54*

9.74

8.99

1.82

12.48*

10.76

Arginine, Arg

Proline, Pro

10.64

1.95

6.10

*Refers to the conjugate acid.

Isoelectronic point, pI
The isoelectronic point or isoionic point is the pH 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 pKas that involve the zwitterion, i.e. that
give the boundaries to its existence.
There are 3 cases to consider....
neutral side chains
These amino acids are characterised by two pKas : pKa1 and pKa2 for the carboxylic
acid and the amine respectively.
The isoelectronic point will be halfway between, or the average of, these two
pKas, 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.

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 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 when the extra
+ve has been neutralised. 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.

1. Introduction
Chemical properties of molecules depend largely on whether they are ionized
or not. Most organic molecules are capable of gaining and/or losing a proton
in aqueous solutions. Proton transfer most frequently occurs between water
and any ionizable atom of the organic molecule. The molecule's response to
protonation or deprotonation depends significantly on the site that was
disturbed by proton transfer. Partial charge distribution in the molecule also
varies with protonation of the acid/base active sites. Since the partial charge
distribution is very sensitive to the protonation-deprotonation process (both
near and far from the disturbed site), it can be used to determine the pK of
a molecule. Our pK prediction program is based on the calculation of partial
charge of atoms in the molecule.
a

2. Definition of Acid Dissociation Constant Ka


Acidic and basic molecules are ionized in aqueous solution. Acidic or basic
character is assigned to the molecule according to Brnsted's rule. The ratio
of the ionized and neutral forms depends on the pH, the temperature and
the ion activity of the bulk phase. The ionization constant K is obtained from
the activity ratio of conjugated base and conjugated acid multiplied with
proton activity.
a

3. Definition of pKa

pK is obtained from the ionization constant of a molecule using the following


definition.
a

When the pH of the solution is equal with pK , the concentrations of dissociated and
undissociated species are equal.
a

4. Definition of the acidic/basic prefix of pKa


definition of the basic prefix
Protonated positive ions are considered as protonated basic sites , therefore ,

"basic" prefix is used for them.


Type of the neutral basic sites are predefined in the pK calculator, they also have
"basic" prefix.
e.g.: if submitted molecule is CH NH or CH NH they get "basic" prefix and
pK =10.08
a

definition of the acidic prefix


Deprotonated negative ions are considered as deprotonated acidic sites , therefore ,
"acidic" prefix is used for them.
Type of the neutral acidic sites are predefined in the pK calculator, they also have
"acidic" prefix.
e.g.: if submitted molecule is CH COOH or CH COO they get "acidic" prefix and
pK =4.54
a

5. Multiprotic Molecules
When a molecule contains more than one ionizable atom, it is called a multiprotic
compound. For these types of molecules we need to distinguish between micro and
macro acidic dissociation constants. The micro acidic dissociation constant is
obtained from the equilibrium concentration of the conjugated acid-base pairs. The
macro acidic dissociation constant is obtained from the global mass and charge
conservation law. When a molecule has N ionizable sites, the total number of micro
species in the solution is 2 .
N

To further understand the difference between micro and macro constants we


consider ionization equilibrium of a triprotic acid AH . Referring to the
different deprotonation sites of the AH molecule we introduce the upper
indexes of the protons.
3

The ionization process of the AH H H molecule in aqueous solution is


described with 12 equilibrium reactions. Micro species and their charge are
summarized in Table 1.
1

6. Ionization Steps of the AH3 Molecule

Table 1. Protonation State of Microspecies


Microspecies

charge

AH H H

AH H , AH H , AH H
1

AH , AH , AH
1

-1
-2
-3

The AH molecule has three macro acidic constants since it is triprotic acid. Macro
acidic constants K , K , and K are obtained from the concentration of the
microspecies:
3

And the three macro pK values of the AH molecule that would be obtained with
routine laboratory measurements are as follows:
a

pK =-log (K )
pK =-log (K )
pK =-log (K )
a,1

10

a,2

10

a,3

10

7. Examples
Example 1.
The ionization steps of p-amino benzoic acid are outlined below. Calculated micro
ionization constants k , k , k and k are indicated on the arrows:
1

Below are the calculated and the experimental pK of p-amino benzoic acid
a

Example 2.
Imides and amides can have either acidic or basic character. The extent of
amide/imide ionization at a given pH is determined by two acid dissociation
constants:
pK assigned to the deprotonation step
a,1

RNH

RN + H
-

and pK assigned to the protonation step


a,2

RNH

+
2

RNH + H

Ratio of anionic and cationic species depends on pK , pK and the pH:


a,1

a,2

If 2pH - (pK + pK ) > 0 than deprotonation of amide/imide is favored and the


molecule is said to have an acidic character.
If 2pH - (pK + pK ) < 0 than protonation of amide/imide is favored and it is
considered to have a basic character.
a,1

a,2

a,1

a,2

Chemists often want to know the ionization state of organic compounds at pH 7.4.
In general, the macro pK s of amide/imide compounds are calculated and their
acidic or basic character determined with the above formulas at pH 7.4.
a

Calculated and measured pK of phtalimide and 2-pyridone are given in Table


a

2.

Table 2. Calculated and Observed Acidity Constants


Compound Calculated pK Observed pK
a

phtalimide

8.22

8.30

2-pyridone

11.40

11.70

Example 3

The value of ionization constants of conjugated acid-base pairs usually falls


between 10 and 10 , so these limits are generally used to predict the pK .
When an ionizable site in the molecule has very weak basic or acidic
character, this can be accomodated by increasing the calculation range.
-10

20

The molecule depicted below contains a very weak basic atom. First, macro
pK is calculated with default limits predefined between 10 and 10 . Then
macro pK is calculated with altered limits defined between 10 and 10 .
Changing of the default settings of macro pK calculation can be done in the
-10

20

-50

20

Tools
Options
pK menu of MarvinSketch.
Calculated and observed acidity constants are summarized in Table 3.
Measured pK s taken from Ref.3.
a

Table 3. Calculated and Observed Acidity Constants


pK

First calc.

Second calc.

Observed

pK

a,1

4.24

4.24

4.51

pK

5.75

5.75

6.01

pK

-35.81

a,2

a,3