1. Experiment Two The amphoteric reaction and the pI determination of protein

2. Aims Master the zwitterionic property of proteins
Master the method to determine the pI （isoelectric point） of proteins

3. Principles: cation zwitterion anion COOH Pr. NH2 COO- COOH COO- Pr.
pH<pI
pH=pI
pH>pI
cation
zwitterion
anion

4. pH>pI pH<pI pH=pI +H+ +OH- +OH- +H+
When the pH=pI，the net charge of protein is zero, lack of identical electric charge repel force，the solubility of protein is minimum, and the proteins are likely to precipitate

5. A series of different pH solutions are prepared and the pH solution that present the maximum precipitation is the corresponding pI of casein.

6. test tubes、droper、scaled-pipette 0.5％ casein-sodium acetate solution
LABORATORY INSTRUMENTS &SOLUTIONS
test tubes、droper、scaled-pipette
0.5％ casein-sodium acetate solution
0.01％ Bromocresol green pH indicator
(pH )

7. 0.02M HCl solution、 0.02M NaOH solution 0.01M acetic acid solution、
LABORATORY INSTRUMENTS &SOLUTIONS
0.02M HCl solution、
0.02M NaOH solution
0.01M acetic acid solution、
0.10M acetic acid solution、
1.00M acetic acid solution

8. PROTOCOL The amphoteric reaction of protein
The determination of the pI of casein

9. observe the color of the solution and why?
PROTOCOL
The amphoteric reaction of protein（1）
prepare a test tube
0.5% casein-sodium acetate solution 10 drops
observe the color of the solution and why?
Bromocresol green pH indicator 3 drops

10. The amphoteric reaction of protein（2）
add 0.02M HCl solution
mix well
observe the color
of the solution
the maximum quantity
of precipitation
add 0.02M HCl solution
observe the color of
the solution and why
mix well
the disappear of the precitation

11. The amphoteric reaction of protein （3）
add 0.02M NaOH solution
mix well
observe the color
of the solution
the maximum quantity
of precitation
add 0.02M NaOH solution
observe the color of
the solution and why
mix well
the disappear of the precitation

12. The amphoteric reaction of protein
Results:
casein-sodium acetate solution
color:blue (pH﹥5.4)

13. [H+] ，sediment
the maximum quantity of sediment(pH=pI)
blue – cyan
（3.8﹤pH﹤5.4）

14. add HCl，[H+] increases，pH＜pI,the precipitate gradually disappear
the moment the precipitate totally disappear
color:yellow(pH﹤3.8)

15. Conclusion 1： Proteins are zwitterion
Proteins are likely to precipitate
at the isoelectric point
The pI of casein locates at between

16. The determination of the pI of casein
prepare 7 test tubes and number them
No. 1 2 3 4 5 6 7
Water
2.4
3.2 —
3.0
1.5
2.75
3.38
1.00M AA
1.6
0.8
0.1M AA
4.0
1.0
0.01M AA
2.5
1.25
0.62 pH
3.5
3.8
4.1
4.7
5.3
5.6
5.9
volumn: mL

17. mix well，then add 20 drops of 0
mix well，then add 20 drops of 0.5% casein-sodium acetate solution to each test tube，then mix immediatelly after each adding。
stay for 30 minutes，mark every test tube with marks(0、+、++、+++、++++ )to indicate the amount of the precipitation，and determine the pI

18. The pI determination of Casein1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7
Precipitates ++
+++
++++ +

19. Conclusion 2： the pH of 4# equals to the pI of casein，i.e.pI=4.7
4# have the most amount of precipitates
the pH of 4# equals to the pI of casein，i.e.pI=4.7

20. Questions: What’s the definition of the isoelectric point of protein?
Why the proteins are likely to precipitate at the isoelectric point?

21. Please hand in your Report in time !
Thank you
Biochemistry laboratory
Changsha Medical University