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TITLE: ISOELECTRIC POINT OF PROTEIN

GROUP: 1B
MEMBERS: MAISARAH BINTI NAZLI
WAFA NABILAH BINTI KAMAL
LIM HUI YING
LIEW JIA-JUNG
NG JIA VEN
LECTURER: DR. AZLINA AHMAD




1.0 INTRODUCTION
Isoelectric point is the pH at which an amphoteric molecule such as protein has a net charge
of zero. It is, however, possible for a protein at the isoelectric point to have localized areas or
patches of positivity or negativity.

2.0 Basic structure of Amino Acids
All the 20 amino acids found in proteins have a carboxyl group (-COOH) and an amino acid
group (-NH
2
) bound to the same carbon atom called the -carbon. Amino acids differ from
each other in their side chains or R-groups, attached to the -carbon which vary in structure,
size and electric charges and therefore influence the solubility of amino acid in water.

2.1 Amino acids have specific acid-base properties
The acid base properties of amino acids depend on the amino and carboxyl groups attached to
the -carbon and on the basic, acidic, or other functional groups represented by R. In the
physiological pH range of 7.35 to 7.45, the carboxyl group of an amino acid is dissociated
and the amino group protonated. This kind of ionized molecule with coexistent negative and
positive charges, is called a dipolar ion or ampholyte. At low pH an anion acid is in its
cationic form with both its amino and carboxyl groups protonated (NH
3
+
and COOH). With a
further increase in pH the amino - NH
3
+
also is deprotonated, resulting in the anionic form of
the molecule. The carboxylic (-COOH) group of an amino acid can donate one proton (H
+
)
and behave as an acid, forming a negatively charged anion. Anion group (-NH
2
) can accept
the proton (H
+
) which behave as a base, forming positively charged cation. Thus, amino
acids in aqueous solution are ionized and act as acids and bases.


2.2 Forms of Amino Acid at Physiological pH
At the pH of blood plasma (7.4) or the intracellular space (7.1) the carboxyl group exists
almost entirely as carboxylate (COO
-
) ion and the amino group is in the protonated form
(NH
3
+
).

2.3 Amphoteric Properties of Amino Acids and Formation of Zwitter Ion at Isoelectric
pH
Substances having a two-way property are called amphoteric or ampholytes (Greek word
ampho means both). As amino acids have both acidic and basic groups, these are amphoteric
in nature.

2.4 Zwitter Ion (Dipolar molecule)
Monoamino monocarboxylic acids exist in aqueous solution as dipolar molecule or zwitter
ions, which means that they have both positive and negative charges on the same amino acids.
The -COOH group is ionized and becomes negatively charged anion (COO
-
) and
The -NH
2
group is protonated to form a positively charged cation (NH
3
+
)
Thus, the overall molecule is electrically neutral. Thus, the molecular species which contain
an equal number of ionizable group of opposite charge and as a result bear no net charge, are
called zwitter ion.
The net charge of an amino acid depends upon the pH of the medium.
At acidic pH, i.e. high concentration of H
+
ions, ionized COO
-
group accept a proton
and becomes uncharged (COOH), so that the overall charge on the molecule is
positive.
At alkaline pH (low concentration of H
+
ions), the NH
3
+
group loses its proton and
becomes uncharged; thus the overall charge on the molecule is negative.

2.5 Isoelectric pH (PI)
The pH at which amino acid bears no net charge and therefore does not move in an electric
field is called isoelectric pH (PI).
By manipulating the pH we can alter the charge on amino acids or proteins, which facilitates
the physical separation of amino acids or proteins. All the ionizable groups present in the
protein will influence the PI of the protein. PI of some proteins is given below:
Pepsin =1.1
Casein = 4.6
Albumin = 4.7
Globulin = 6.4
At pH values above or below the isoelectric pH, they carry a net negative or positive charge
and migrate to anode (positively charged electrode) or cathode (negatively charged electrode).

2.6 Applications of Isoelectric Point (PI)
The stability of the protein in solution depends on the charge and hydration of the protein
molecule. The factors which neutralize the charge or remove water of hydration will cause
precipitation of proteins. One of the factors used for precipitation is isoelectric pH. All
proteins are least soluble at their isoelectric pH (PI) and can then be precipitated.






References
1. Biochemistry and Molecular Biology, William H. Elliott & Daphne C. Elliott
2. Biochemistry, Linpincott illustrated reviews 5
th
edition, Harvey & Ferrier
3. www.clevernotes.ie
4. http://en.wikipedia.org/wiki/Amino_acid