Experiment Two The amphoteric reaction and the pI determination of protein

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

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

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

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

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 (pH3.8-5.4)

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

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

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

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

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

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

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

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

Conclusion 1： Proteins are zwitterion Proteins are likely to precipitate at the isoelectric point The pI of casein locates at between 3.8-5.4

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

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

The pI determination of Casein 1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7 Precipitates ++ +++ ++++ +

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

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

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