Determination of the Isoelectric

Points of Casein
• Due to its amphoteric nature, the solubilty of
casein depends greatly on the PH of the
medium.
 Determination of the Isoelectric
Points of Casein
• Certain proteins e.g. gelatin are relatively
soluble in water even at their I.E.P.
BUT maximum precipitation can be obtained at
the the isoelectric point by addition of some
reagents such as, ethanol which dehydrates the
molecule and allow neutralization of charge.
* Principle
• Using acetate buffer of different PH values the
approximate position of the isoelectric point of
casein can be obtained by determining the PH
of minimum solubility.
• The PH of any solution can be calculated from
Handerson-hasselbalch equation.
PH = Pka + log [Sodium acetate]
[acetic acid]
 Tube # 1 2 3 4 5 6 7

0.05% casein solution

in 0.1ml CH3COONa 1 1 1 1 1 1 1
(ml)

Distilled Water (ml) 8.4 8 8.8 8.5 8 7 7.4

0.01M acetic acid (ml) 0.6 1 - - - - -

0.1M acetic acid (ml) - - 0.2 0.5 1 2 -

1M acetic acid (ml) - - - - - - 1.6

* Results
Tube # ppt at 0 ppt at 10 ppt at 30 PH
min min min
1
2
3
4
5
6
7
* Results
PK acetic acid = 4.5

PH = PKa + log [CH3COONa]

[CH3COOH]

Samar A. Damiati