B.

DIGESTION IN THE STOMACH

1. Peptic Digestion:

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

INTRODUCTION:

METHODOLOGY:

1. PREPARE TEST TUBES ACCORDING TO THE PLAN PRESENTED. 2. SHAKE EACH TUBE AND DETERMINE THE PH USING PH PAPER.

Test tube Number Neutral Pepsin, 2% ml HCL, 0.01 N HCL, 0.05 N Lactic Acid, 4 N Na2CO3, 0.5 N ml ml ml ml

1 5 _ _ _ _ *

2 _ 5 _ _ _ *

3 4 _ 1 _ _ *

Hard-boiled egg white* (*equal slices, cu.cm)

Test tube Number Neutral Pepsin, 2% ml HCL, 0.01 N ml HCL, 0.05 N ml Lactic Acid, 4 N ml Na2CO3, 0.5 N ml Hard-boiled egg white* (*equal slices, cu.cm)

4 4 _ _ 1 _ *

5 4 _ _ _ 1 *

Test

tube Number 1
5)

(Neutral Pepsin, 2% ml

Slightly Acidic

Test

tube Number 2
(HCL, 0.01 N ml 5)

Acidic

Test

tube Number 3
(HCL, 0.05 N ml 1)

Acidic

Test

tube Number 4 Acidic

(Neutral Pepsin, 2% ml 4) (Lactic Acid, 4 N ml 1)

Test

tube Number 5
1)

(Neutral Pepsin, 2% ml 4) (Na2CO3, 0.5 N ml

Basic

 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 of the hard-boiled egg white.

Determine the extent of digestion visually by noting the size of the hardboiled egg white.

TEST TUBE NUMBER

NO REACTION NO VISIBLE CHANGE IN SIZE

TEST TUBE NUMBER

THERE IS VISIBLE CHANGE IN SIZE. SLIGHT ENLARGEMENT OF THE HARD BOILED EGG WHITE

TEST TUBE NUMBER 3

THERE IS VISIBLE CHANGE IN SIZE. SLIGHT ENLARGEMENT OF THE HARD BOILED EGG WHITE

TEST TUBE NUMBER 4

THERE IS VISIBLE CHANGE IN SIZE. SLIGHT ENLARGEMENT OF THE HARD BOILED EGG WHITE

TEST TUBE NUMBER 5

NO REACTION NO VISIBLE CHANGE IN SIZE

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.

RESULTS:

TEST TUBE NUMBER 1

TEST TUBE NUMBER 2

TEST TUBE NUMBER 3

TEST TUBE NUMBER 4

TEST TUBE NUMBER 5

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 shortchain polypeptides (it will not cleave on all peptide chains)

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: 1. WHAT ARE THE OPTIMUM CONDITIONS FOR THE ACTIVITY OF PEPSIN? RENNIN? Pepsin is most active in acidic environments between 37C and 42C.Accordingly, its primary site of synthesis and activity is the stomach (pH 1.5 to 2). Pepsin exhibits maximal activity at pH 2.0.

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 necessary because sometimes the stomach is diluted by outside materials.

2. DIFFERENTIATE INFANT CURDLING FROM ADULT 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 is catalyzed by hydrochloric acid and pepsin.

3. WHY IS THERE NO DIGESTION OF PROTEINS IN GASTRIC JUICE WITH PH ABOVE 7?

The digestive power of pepsin is greatest at the acidity of 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.

4. WHAT SUBSTANCES OTHER THAN HYDROCHLORIC ACID CONTRIBUTE TO THE ACIDITY OF GASTRIC CONTENTS?

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.