生物化学与分子生物学教研室 experiment two the amphoteric reaction and the pi...

生物化学与分子生物学教研室

Experiment TwoThe amphoteric

reaction and the pI determination of

protein


Master the zwitterionic property of proteins

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

Aims


Pr.COOH

NH2

Pr. COO-

NH3+

Pr. COO-

NH2Pr.COOH

NH3+

pH=pI

+OH-

pH>pI

+H+

+OH-

+H+

pH<pI

zwitterion cation anion

Principles:


+OH-

+H++OH-

+H+

pH<pI pH>pIpH=pI

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.


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 、 0.10M acetic acid solution 、 1.00M acetic acid solution

LABORATORY INSTRUMENTS &SOLUTIONS


• The amphoteric reaction of protein

• The determination of the pI of casein

PROTOCOL


• The amphoteric reaction of protein （ 1 ）

PROTOCOL

observe the color of the solution and wh

y?

prepare a test tube

0.5% casein-sodium acetate solution 10 drops

Bromocresol green pH indicator 3 drops



add 0.02M HCl solution

mix well

the maximum quantity of precipitation

the disappear of the precitation

add 0.02M HCl solution

mix well

observe the color of the solution

observe the color of the solution and why



add 0.02M NaOH solution

mix well

the maximum quantity of precitation

the disappear of the precitation

add 0.02M NaOH solution

mix well

observe the color of the solution

observe the color of the solution and why


The amphoteric reaction of protein

Results: casein-sodium acetate s

olution 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 ：

The pI of casein locates at between 3.8-5.4

Proteins are likely to precipitateat the isoelectric point

Proteins are zwitterion


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

• The determination of the pI of casein

volumn: mL


mix well ， then add 20 drops of 0.5% casei

n-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 ， a

nd determine the pI


1 2 3 4 5 6 7Precipitat

es0 ++ +++ ++++ ++ + 0

The pI determination of Casein

1 2 3 4 5 6 71 2 3 4 5 6 7


Conclusion 2 ：

4# have the most amount of precipitates

the pH of 4# equals to the pI of

casein ， i.e.pI=4.7


What’s the definition of the isoelectric point of protein?

Why the proteins are likely to precipitate at the isoelectric point?

Questions:


Please hand in your Report in time !

Thank youBiochemistry laboratory

Changsha Medical University

生物化学与分子生物学教研室 experiment two the amphoteric reaction and the pi...

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