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7/28/2019 Ppt Biochem
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B. DIGESTION IN THE STOMACH
1. Peptic Digestion:
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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
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INTRODUCTION:
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METHODOLOGY:
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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)
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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)
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Test tube Number 1
(Neutral Pepsin, 2% ml 5 )
Slightly Acidic
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Test tube Number 2(HCL, 0.01 N ml 5)
Acidic
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Test tube Number 3(HCL, 0.05 N ml 1)
Acidic
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Test tube Number 4 (Neutral Pepsin, 2% ml 4)
(Lactic Acid, 4 N ml 1)
Acidic
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Test tube Number 5 (Neutral Pepsin, 2% ml 4)
(Na2CO3, 0.5 N ml 1)
Basic
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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.
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TESTTUBE NUMBER 1
NO REACTION
NO VISIBLE CHANGE IN SIZE
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TESTTUBE NUMBER 2
THERE IS VISIBLE CHANGE IN SIZE.
SLIGHT ENLARGEMENT OF THE HARD BOILED
EGG WHITE
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TESTTUBE NUMBER 3
THERE IS VISIBLE CHANGE IN SIZE.
SLIGHT ENLARGEMENT OF THE HARD BOILED
EGG WHITE
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TESTTUBE NUMBER 4
THERE IS VISIBLE CHANGE IN SIZE.
SLIGHT ENLARGEMENT OF THE HARD BOILED
EGG WHITE
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TESTTUBE NUMBER 5
NO REACTION
NO VISIBLE CHANGE IN SIZE
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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.
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RESULTS:
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TESTTUBE NUMBER 1
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TESTTUBE NUMBER 2
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TESTTUBE NUMBER 3
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TESTTUBE NUMBER 4
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TESTTUBE NUMBER 5
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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)
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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
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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.
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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
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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
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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.
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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.