Properties of Protein

Merlyn C. Cruz, MSc, RMT, RM

Casein
! Name for a family of related Phosphoproteins
! Makes up 80% of the proteins in cows milk
and between 20%-45% in human milk
! The most important protein in milk which
supplies essential amino acids as well as
carbohydrates, Ca and P
! Functions as storage protein which consists
high number of non-interacting proline
peptides
! With relatively weaker tertiary structure, it
can not denature
! Relatively hydrophobic
! Has a negative charge in milk (milks
pH=6.6)
! Can be isolated from milk by acidification to
bring it to its isoelectric point
Isolation of Casein by Acidification
! Proteins are least soluble in water at their isoelectric
points

! At pH higher or lower than its pI, they tend to

aggregate by electrostatic interaction and the aggregates
precipitate out of the solution
Important Steps During Isolation
!Precipitation with 10% acetic acid lowers
the pH of milk to bring casein to its pI
!Addition of ethyl alchol-ethyl acetate
mixture and filtration dissolves and
removes any fats that may have co-
precipitated with casein
! Drying removes the water obtained upon
washing of casein to be able to determine the
% (m/m) of casein in the milk sample
! Addition of NaOH & H2O
- neutralizes the acetic acid and redissolves
at least some of the proteins
- brings the pH back above the pI of the
protein
 Qualitative Determination Tests for
Protein
! Biuret Test general test for proteins
! when protein reacts with copper(II) sulfate, a positive
test is the formation of a copper complex which has a
violet color
! Ninhydrin Test Amino acids with free NH2 group
and proteins containing free amino groups react with
ninhydrin to give a purple-blue complex
 Determination of the pI of Casein
pI :
! the pH at which the majority of molecules of a
compound in solution have no net charge

At neutral
pH, the COOH
donates its H + to - NH (internal
2
acid-base reaction)

Zwitterion molecule has positive

charge on one atom and negative
charge on another atom, but
which, no net charge
! Amino acids in solution exist in three different
species (zwitterions, positive ion, and negative
ion)
! Equilibrium shifts with change in pH

COOH COO- COO-

+H N +H N C H H2N C H
3 C H 3

CH3 CH3 CH3

Low pH High pH
Zwitter Ion (net - charge)
(net + charge) (net neutral charge)
 + -
! Upon addition of an acid, H3O protonates the -COO
! Upon addition of a base, the -NH3+ is
deprotonated to donate to the -OH- from the
base
 Thus, the over-all equation is:
pH of milk : 6.6
! This pH is above the pI of casein, thus, at this pH, casein
has a net negative charge
! This results to an electrostatic attraction between casein
proteins in milk
! The aggregates of proteins is observed as precipitate

! Addition of acetic acid lowers the pH of the milk
! Casein has a pI lower than the pH of milk, where it
has no net charged
! At neutral charge, no electrostatic attraction occurs
between proteins, thus, no precipitate is formed

! pH at which no precipitation occurs : ~ pH 4 5

! pI of casein : pH 4.6
 Uses of Casein
! Paint - casein paint is a fast drying, water-soluble medium
used by ancient artists until the late 60s and became less
popular with the advent of acrylic paint
! Glue casein based-glues were popular for woodworks
! Cheesemaking cheese consists of proteins and fats from
milk and is produced by coagulation of casein
! Protein supplements- can form a gel and clot in the
stomach which provides a sustained slow release of amino
acids into the blood stream
! Medical and Dental Uses casein derived compounds are
used in tooth remineralization products to stabilize
amorphous calcium phosphate