Ppt Biochem

7/28/2019 Ppt Biochem

1/50

B. DIGESTION IN THE STOMACH

1. Peptic Digestion:


2/50

OBJECTIVES:

1. To determine the optimum conditions for the

digestive enzymes;

2. To investigate the activity of these digestive

enzymes

3. To illustrate their efficiency; and

4. To perform methods that will determine

enzymatic activity


3/50

INTRODUCTION:


4/50

METHODOLOGY:


5/50


6/50

1. PREPARETESTTUBESACCORDINGTOTHEPLANPRESENTED.

2. SHAKEEACHTUBEANDDETERMINETHEPH USINGPH PAPER.

Test tube Number 1 2 3

Neutral Pepsin, 2% ml 5 _ 4

HCL, 0.01 N ml _ 5 _ HCL, 0.05 N ml _ _ 1

Lactic Acid, 4 N ml _ _ _

Na2CO3, 0.5 N ml _ _ _

Hard-boiled egg white* * * *

(*equal slices, cu.cm)


7/50

Test tube Number 4 5

Neutral Pepsin, 2% ml 4 4

HCL, 0.01 N ml _ _

HCL, 0.05 N ml _ _

Lactic Acid, 4 N ml 1 _

Na2CO3, 0.5 N ml _ 1

Hard-boiled egg white* * *

(*equal slices, cu.cm)


8/50


9/50

Test tube Number 1

(Neutral Pepsin, 2% ml 5 )

Slightly Acidic


10/50


11/50


12/50

Test tube Number 2(HCL, 0.01 N ml 5)

Acidic


13/50


14/50


15/50

Test tube Number 3(HCL, 0.05 N ml 1)

Acidic


16/50


17/50


18/50

Test tube Number 4 (Neutral Pepsin, 2% ml 4)

(Lactic Acid, 4 N ml 1)

Acidic


19/50


20/50


21/50

Test tube Number 5 (Neutral Pepsin, 2% ml 4)

(Na2CO3, 0.5 N ml 1)

Basic


22/50


23/50


24/50


25/50

3. Add equal slices of hard-boiled egg

white to each tube.

4. Add 4 drops of toluene.

5. Cover and incubate in your locker till

the next laboratory meeting. Avoid

shaking the tubes after the addition ofthe hard-boiled egg white.

Determine the extent of digestionvisually by noting the size of the hard-

boiled egg white.


26/50


27/50

TESTTUBE NUMBER 1

NO REACTION

NO VISIBLE CHANGE IN SIZE


28/50


29/50

TESTTUBE NUMBER 2

THERE IS VISIBLE CHANGE IN SIZE.

SLIGHT ENLARGEMENT OF THE HARD BOILED

EGG WHITE


30/50


31/50

TESTTUBE NUMBER 3



EGG WHITE


32/50


33/50

TESTTUBE NUMBER 4



EGG WHITE


34/50


35/50

TESTTUBE NUMBER 5

NO REACTION

NO VISIBLE CHANGE IN SIZE


36/50


37/50

6. Determine the extent of digestion by means of

the Biuret test as follows. Shake the tubes, let stand and withdraw 1 ml of the

supernatant liquid.

Neutralize those from the test tubes no. 2, 3 and 4

with solid NA2CO3 first. To each 1 ml supernatant liquid, add 1 ml of 10%

NaOH and 2 drops of 1% CuSO4.

7. Mix well and observe any resulting color.


38/50

RESULTS:


39/50

TESTTUBE NUMBER 1


40/50

TESTTUBE NUMBER 2


41/50

TESTTUBE NUMBER 3


42/50

TESTTUBE NUMBER 4


43/50

TESTTUBE NUMBER 5


44/50

BIURET TEST:

A chemical test used to detect the presence

of peptide bonds.

(+) test = purple (presence of proteins)

() test = blue to pink when combined with short-

chain polypeptides (it will not cleave on all peptide

chains)


45/50

If there is no digestion yet, incubate longer.

Compare colors obtained in each test tube with that

given by 0.5%peptone (Pinkish solution).

NO AVAILABLE 0.5% PEPTONE

QUESTIONS


46/50

QUESTIONS:

1. WHATARETHEOPTIMUMCONDITIONSFORTHEACTIVITY

OFPEPSIN? RENNIN?

Pepsin is most active in acidic environments

between 37C and 42C.Accordingly, its primary

site of synthesis and activity is the stomach (pH 1.5to 2). Pepsin exhibits maximal activity at pH 2.0.


47/50

Optimal pH for rennin is around 6.0-6.5, as this

closely resembles the acidic environment of the

stomach where rennin is most commonly found.

However rennin also works at neutral pH, although

at a much lower level of reaction. This is necessarybecause sometimes the stomach is diluted by

outside materials.

2 DIFFERENTIATE INFANT CURDLING FROM ADULT


48/50

2. DIFFERENTIATEINFANTCURDLINGFROMADULT

CURDLING.

Infant curdling is catalyzed by the enzyme rennin

acting on the hydrolysis of milk protein Casein.

Calcium paracasenate is necessary in the process.

On the other hand, this enzyme is said to be absent

in the stomach of adults. In this case, curdling iscatalyzed by hydrochloric acid and pepsin.

3 WHY IS THERE NO DIGESTION OF PROTEINS IN


49/50

3. WHYISTHERENODIGESTIONOFPROTEINSIN

GASTRICJUICEWITHPHABOVE 7?

The digestive power of pepsin is greatest at the acidityof normal gastric juice (pH 1.52.5). In the intestine the

gastric acids are neutralized (pH 7), and pepsin is no

longer effective. At pH 5.0 activity begins to decline as

the pepsin protein denatures and is inactive at pH 6.5

and above, however pepsin is not fully denatured or

irreversibly inactivated until pH 8.0.


50/50

4. WHATSUBSTANCESOTHERTHANHYDROCHLORICACID

CONTRIBUTETOTHEACIDITYOFGASTRICCONTENTS?

Hydrogen ions contribute to the acidity of the

gastric contents. Lactic acid produced by

fermentation is another contributing factor. Aside

from, the stomach also consists of large quantities

of potassium chloride (KCl) and sodium chloride

(NaCl). There are no exact proportions between

the different acid types, resulting in the pH

fluctuation.

Documents

Ppt Biochem