pH and Protein Solubility
Materials:
Casein
2M HCl
2M NaOH
Analytical balance
Magnetic stirrer and Stirring bar
DI water
0.1M NaOH
600mL Beaker or Flask
2mL Pipets, graduated
Safety:
Wear gloves and goggles
Procedures:
Initial Casein Solution:
 Add 25mL of 0.1M NaOH and stirring bar to a 600ml beaker or flask on a
magnetic stirrer
 Add 225mL of DI water and stir at moderate speed
 Add 1g of casein and stir to dissolve (soln. will be slightly cloudy)
 Insert pH probe in solution and record pH value
Casein Solution after HCl:
 With rapid stirring add 2M HCl in 0.5mL increments using a graduated pipet
 After 1-2mL of acid has been added, the cloudiness will reach a maximum
(Isoelectric Point pH at isoelectric point is 4-5)
 Insert pH probe in the solution and record pH value
 Continue adding 2M HCl in 0.5mL increments until solution is clear again
(Cloudiness will fade and ppt. will redissolve after addition of 2-3mL of acid,
when the pH drops below the isoelectric point, pH≤ 2)
Casein Solution after NaOH:
 Continue to stir the soln. reverse the process by adding 2M NaOH in 0.5mL
increments using a clean graduated pipet
 After 2-3mL has been added protein will ppt. out again (isoelectric point)
 Continue adding NaOH in 0.5mL increments with stirring until soln. is clear again
(soln. will clear after additional 1-2 mL of NaOH, pH > 10-12)
Discussion:
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Casein is principal protein in milk (80% of total protein content)
Casein is a phosphoprotein (it contains a large # phosphate groups attached to
amino acid side chains in its polypeptide structure)
Negatively charged phosphate groups are balanced by positively charged calcium
ions and are responsible for the high nutritional calcium content in milk
Casein is almost completely insoluble in water at neutral pH=7
Casein, like other proteins, is an ionic species containing amino groups and
carboxyl groups on its terminal amino acid
It also contains a variety of other acidic and basic groups on the side chains of its
non-terminal amino acids.
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The effect of pH on the solubility of casein reflects the ionization of the acidic
and basic groups in its structure
At high pH:
 casein will have net negative charge due to ionization of all acidic side chains
(CO2-) in its structure
 B/c casein is ionized at high pH values; it is soluble in dilute NaOH soln.
At low pH:
 casein will have a net positive charge due to the protonation of all basic side
chains (NH3+) in its structure
 B/c casein is ionized at low pH values; it is also soluble in strongly acidic soln.
At intermediate pH:
 casein will contain roughly equal numbers of positively and negatively charged
groups
 protein will have a net charge of zero
 casein is insoluble in neutral soln. b/c it is not charged under these conditions
 solubility of protein is usually minimum at its isoelectric point
 Isoelectric point: pH at which a protein has a net charge of zero for casein do to
the attached phosphate groups the isoelectric point is close to pH=4
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