KAU-Faculty of Science- Biochemistry department

Analytical biochemistry lab (Bioc 343) 2012

T.A Nouf Alshareef
nf.shareef@hotmail.com
 Isoelectric point (pI)
Isoelectric point (pI):
• is pH at which amino acid or protein has no net charge (zero)
• Solubility at pI is minimum (i.e. protein precipitate)
• amino acid at this point is zwitter ion.

Important of pI:
• We can separate protein according to pI (salting in/salting out).
• We can use it in separation method as:
 ion exchange chromatography choose st.phase
 protein electrophoresis choose buffer pH
• Proteins behave as poly-ionic macromolecules
• Amino acids are amphoteric molecules: (contains at least one
amino and one carboxyl group, and they have acid-base properties)

when reacts with acids, it acts as a base:

R Acid media R
+ +
H3 N C COO + H3O H3 N C COOH
H H + H2 O

Zwitterio Acid Cationic

when reacts with bases and acts as acid:

R base media R
+
H3 N C COO + OH H2 N C CO O
H H + H2 O

Zwitterio Base Anionic form

• Since a-carboxyl and a-amino groups are weak acids and bases, respectively,
buffering action by these groups will occur.
• Proteins can be separated according to their pI in a process known
as isoelectric focusing.
 at pH below pI proteins carry a net positive charge.
 at pH Above pI proteins carry a net negative charge.

• pH of electrophoresis gel is determined by buffer used for that gel:

- If pH of buffer is above pI of protein >>>protein will migrate to +ve pole
(Negative charge is attracted to positive pole)

- If pH of buffer is below pI >>>>>protein will migrate to –ve pole of gel

(Positive charge is attracted to negative pole).

- If protein is run with a buffer pH that is equal to pI, it will not migrate.

• This also true for individual amino acids.

 Titration curve
• To determine pI of amino acid or protein, we will titrate it with
base and then with acid

• A curve will be drown between pH and volume of acid or base.

• From the curve we can determine:

- pKa
- pKb
• So,
pI = halfway between two points of strongest buffering capacity.

PI = (pK1 + pK2)
2
The isoelectric point (pI) can be estimated by:
PI = (pK1 + pK2) K1: dissociation constants of carboxyl group
K2: dissociation constants of amino groups
2
• Titration curve is very useful in identification of amino acid .

Example: glycine has two dissociation steps:

loss of H+ from the acidic carboxyl group at low pH
loss of H+ from the more basic amino group at high pH

• pKa value for each dissociable group of an amino acid can be

determined from a titration curve as :

pKa: is the midpoint of plateau in each buffering region in

titration curve.
Procedure:
• 20 ml of your selected amino acid
solution into the a beaker.

• Assemble:
titration stand, burette and pH meter.

• Record the initial pH of the amino acid

solution and the initial reading of your
burette (Which should be 0.0 ml)
• Turn on the stirrer, check that the magnetic bar does not collide
with the electrodes.

Base titration:
• Titrate with base by dropwise addition of the 0.5 N NaOH.
• Record the volume addition of NaOH and pH of the solution
throughout the titration until a pH > 12 is reached.

Acid titration:
• Repeat the titration with 0.25 N H2SO4.
• Record the volume of added H2SO4 and pH of the solution
throughout the titration until a pH below 1.5 is reached.
 Result sheet
Data Collection:
Initial pH reading _______ Volume added _0.0 ml_

H2SO4 NaOH
ml pH ml pH
added added
0.2 0.2
0.4 0.4
0.6 0.6
1.0 0.8
2.0 1.0
3.0 2.0
3.0
4.0

End End
point point
(1.5) (12.00)
Answer the following questions:

• Plot pH versus ml of NaOH and H2SO4 solutions added.

• Use pH as the dependent variable (Y-axis) and ml of NaOH or
H2SO4 as the independent variable (X-axis)

Determine the pI of your amino acid from curve and by

calculation?