What is the isoelectric point?

Explain a protein purification

method based on pI.

Isoelectric point
From Wikipedia, the free encyclopedia
The isoelectric point (pI) is the pH at which a particular molecule or
surface carries no net electrical charge.Amphoteric molecules
called zwitterions contain both positive and negative charges depending on
thefunctional groups present in the molecule. They are affected by pH of their
surrounding environment and can become more positively or negatively charged
due to the loss or gain of protons (H+).
The pI value can also affect the solubility of a molecule at a given pH. Such
molecules have minimum solubility in water or salt solutions at the pH which
corresponds to their pI and often precipitate out of solution. Biological
amphoteric molecules such as proteins contain both acidic and basic functional
groups. Amino acids which make up proteins may be positive, negative, neutral
or polar in nature, and together give a protein its overall charge. At a pH below
their pI, proteins carry a net positive charge; above their pI they carry a net
negative charge. Proteins can thus be separated according to their isoelectric
point (overall charge) on a polyacrylamide gelusing a technique called isoelectric
focusing, which utilizes a pH gradient to separate proteins. Isoelectric focusing is
also the first step in 2-D gel polyacrylamide gel electrophoresis.

Calculating pI values
For an amino acid with only one amine and one carboxyl group, the pI can be
calculated from the pKa's of this molecule.

For amino acids with more than two ionizable groups, such as lysine, the
same formula is used, but this time the two pKa's used are those of the two
groups that lose and gain a charge from the neutral form of the amino
acid.Lysine has a single carboxylic pKa and two amine pKa values (one of which
is on the R-group), so fully protonated lysine has a +2 net charge. To get a
neutral charge, we must deprotonate the lysine twice , and therefore use theR-
group and amine pKa values (found at List of standard amino acids).

However, a more exact treatment of this requires

advanced acid/base knowledge and calculations.
The pH of an electrophoretic gel is determined by the buffer used for that gel. If
the pH of the buffer is above the pI of the protein being run, the protein will
migrate to the positive pole (negative charge is attracted to a positive pole). If
the pH of the buffer is below the pI of the protein being run, the protein will
migrate to the negative pole of the gel (positive charge is attracted to the
negative pole). If the protein is run with a buffer pH that is equal to the pI, it will
not migrate at all. This is also true for individual amino acids.

What are the different ways in which amino acids can be classified? Illustrate
with examples.

Figure 1. A Venn diagram showing the relationship of the 20 naturally occurring amino acids to a
selection of physio-chemical properties thought to be important in the determination of protein
structure

Classification of Amino Acids

There are twenty amino acids that are used to form proteins in the human body, these are called
the proteinogenic2 amino acids. There appear to be many different classification systems, three
of which are presented here.

Timberlake3, classifies the amino acids using the system presented in Table 1. She uses a simple
method of classification, identifying amino acids as polar or non-polar. A further subclassification
of acidic-polar when the side chain contains a carboxylic acid, and basic-polar when the side
chain contains an amino group is also introduced.

Classification Amino Acid

Glycine

Alanine

Valine

Leucine

Nonpolar Isoleucine

Proline

Methionine

Phenylalanine

Tryptophan

Serine

Threonine

Asparagine
Polar
Glutamine

Cysteine

Tyrosine

Aspartic Acid
Acidic (Polar)
Glutamic Acid

Lysine

Basic (Polar) Arginine

Histidine
 Table 1 Classification of amino acids (after Timberlake3)

Devlin4 classifies amino acids along structural lines. Devlin’s system is presented in Table 2.

Superstructure Structure Amino Acid

Glycine
Monoamino,
moncarboxylic
L-Alanine

L-Valine

Unsubstituted L-Leucine

L-Isoleucine

L-Proline
Heterocyclic
L-Phenylalanine

L-Tyrosine
Aromatic
L-Tryptophan

Thioether L-Methionine

L-Serine
Hydroxy
L-Threonine

Mercapto L-Cysteine

L-Asparagine
Carboxamide
L-Glutamine

L-Aspartate
Monamino, dicarboxylic
L-Glutamate

Diamino, monocarboxylic L-Lysine

 L-Arginine

L-Histidine

Table 2 Classification of amino acids (after Devlin4)

A third method of classification, sourced from Koolman2 is presented in Table 3. This

classification system is again based on structure of the side chain.

Classification Amino Acid

Glycine

Alanine

Alphatic (do not contain N,O,S in side

Valine
chain)

Leucine

Isoleucine

Cysteine
Sulfur-containing
Methionine

Phenylalanine

Aromatic (benzene ring in side chain) Tyrosine

Tryptophan

Serine

Threonine
Neutral (hydroxyl or amide groups in
side chain)
Asparagine

Glutamine

Acidic (carboxylate groups in side Aspartic acid

chain)
 Glutamic acid

Lysine
Basic
Arginine

Imino acid (special case) Proline

Table 3 Classification of amino acids (after Koolman2)