BIOCHEM LAB - Test Tube 3 would also have a delayed

decolorization because formalin wasn’t

Experiment 5
added but water was added
Enzymes: Nature and Specificity - All three test tubes were heated to
deactivate xanthine oxidase
Catalase-peroxidase - Test Tube 2 should have the fastest
- enzymes that can be extracted from time to be decolorized
potatoes TT. No. Time to decolorize
- classified as oxidoreductase 1 slow
- present in the blood, liver, and in kidney 2 fast
as free radicals (detoxifies the body) 3 slow

Biuret Test
Test for Salivary Amylase
- test for presence of catalase
- general test for proteins because it - Lugol’s Solution (Iodine) is the indicator;
detects peptide bonds blue-black is the positive result
- violet is the positive result - Salivary amylase (Ptyalin) hydrolyzes α-
glycosidic bonds (specifically α-1,4
Test for Catalase activity glycosidic bonds)
- formation of bubbles means that there is - Starch is classified as polysaccharide
catalase activity; more bubble formation which has α-1,4 glycosidic bonds
= more presence of the enzyme therefore will be hydrolyzed by Salivary
- Benzidine is an enzyme inhibitor; Amylase to Dextrin and Maltose
identifies blood in crime scenes (subsequently to Glucose)
(catalase + benzidine = blue-green - Dextrin and Maltose are disaccharides
color) - Phosphate buffer is added to maintain
- blue-green is the positive result the pH suitable for the salivary amylase
- Theoretically, there SHOULDN’T be a
Test for oxidase activity in milk blue-black result in the first test tube
since the starch should have been
- Xanthine Oxidase aka Schardinger’s
catalyzed by the salivary amylase; the
enzyme catalyzes the oxidation of
color should be yellow or orange
aldehydes and acids in an anaerobic
- Heating is done to denature the salivary
environment
amylase; the result in the spot plate
- Methylene blue is reduced to colorless
should be blue first because the salivary
‘leuko form’ when catalyzed by xanthine
amylase is denatured then yellow as
oxidase
time goes by
- Formalin helps in maintaining the pH
environment to make xanthine oxidase
efficient in its activity
- Mineral oil prevents access to air to
make the environment anaerobic
- Heat is applied to increase the reaction;
Test Tube 1 would have a delayed
decolorization because xanthine oxidase
was deactivated thus not being able to
catalyze methylene blue to leuko form
due to the application of heat
Experiment 6 Effect of Co-enzymes
Factors Affecting Enzyme Activity - Yeast is used because it has a lot of
enzymes; eg: sucrase (invertase),
Effect of Temperature
zymase, maltase, and reductase
TT Temperature Theoretical - Sucrase in yeast can hydrolyze sucrose
No. Result to glucose and fructose
1 40° C yellow - All of monosaccharides (glucose,
2 60° C blue-black fructose, galactose) are reducing sugars
3 10° C blue-black - Positive result for Benedict’s test (used
T1 – optimal temperature is 37° C T3 – enzyme is deactivated
to test for the presence of reducing
T2 – enzyme is deactivated sugars) is brick red which means
Note: Food spoilage is prevented by freezers sucrose is catalyzed by sucrase
because the low temperature deactivates the - Main enzyme is in the supernatant while
activity of microorganisms and bacteria the co-enzyme is in the residue
TT Reagents Benedict’s
No. Test
Effect of pH 1 sucrose + H2O -
2 sucrose + supernatant +
Pepsin – secreted in stomach; acidic (works
3 sucrose + H2O + +
best in acidic environment) residue
Pancreatin – secreted in the pancreas, 4 sucrose + supernatant +
released in small intestine; basic (works best in + residue
T1 – only water, no enzyme T3 – enzyme is in residue
basic environment)
T2 – enzyme is in supernatant T4 – enzyme is in supernatant
Proteases – acts on proteins (albumin is added and and residue

to test the activity of the enzymes)

TT Best Digestion Biuret
No. environment of Test
for enzyme? albumin
1 YES High violet
2 NO Low violet
3 NO Low violet
4 YES High violet
TT Reagents
No.
1 pepsin +
HCl
2 pepsin +
Na2CO3
3 pancreatin
+ HCl
4 pancreatin
+ Na2CO3
Experiment 7
Nucleic Acids
Test for purines
Solubility of RNA
- Ammonium phosphomolybdate will
Environment Solubility cause the hydrolysis of glycosidic bonds
Cold water slightly soluble - Two bonds in purines: (1) glycosidic
Hot water soluble bonds (2) phosphodiester bonds
dil. HCl soluble - Positive result: foamy white gelatinous
dil. NaOH soluble precipitate
Alcohol insoluble

Basic Components of Nucleic Acid

- Phosphate
- Nitrogenous Bases
- Ribose
Test for Sugar
- Ribose is a monosaccharide which is
classified as pentose (sugar portion of
RNA)
- Molisch test is the general test for
carbohydrates; positive result is violet
Test for protein
- Nucleic acids have amino residues,
therefore technically it has proteins
- Biuret test is utilized; positive result is
violet
Test for phosphate
- Sample will yield a yellow solution
because of ammonium
phosphomolybdate
EXPERIMENT 5
Introduction
Enzymes are biological catalysts produced by living tissues that increase the rate of reactions and only
require lower activation energy as compared to inorganic catalyst. All known enzymes are proteins (globular)
and mostly have a tertiary type of protein structure. Some enzymes are simple proteins such as pepsin, trypsin
etc. which are composed of only amino acid chains or peptides. Other enzymes need some non-protein chemical
components for their proper biochemical functioning. They are called Holoenzymes or conjugated enzyme
which is composed of protein and non-protein portions. The protein portion is called “apoenzyme” while the
non-protein portion component is called co-factors. Co-factors could be a metal ion activator such as Zn+1, Mn+2,
Mg+2, Fe+2, Cu+2. Other co-factors are co-enzymes such as FAD, FMN and NAD.
Enzymes exhibit specificity of action because they have a specific active site, where the enzyme binds a
substrate/s and catalyzes the reaction. The substance acted upon by the enzyme is called a substrate.

Different enzymes have different specificity of action such as:

1. Absolute specificity. The enzyme catalyzes a specific reaction for only a specific substrate and has no
catalytic effect on closely associated substrate.
2. Stereochemical specificity. The enzyme shows specificity for only one stereoisomeric form of the
substrate.
3. Group specificity. The enzyme is less selective and acts upon structurally similar molecules that have
the same functional group.
4. Linkage specificity. This enzyme is the least specific of all and attacks a particular kind of chemical
bond.

Enzymes can be classified according to the reaction they catalyze. Examples include oxidoreductase which
catalyzes oxidation reduction reactions; transferase which facilitates the transfer of a group such as methyl,
amino or acetyl from one molecule to the other. Catalase present in potatoes is an example of an oxidoreductase
facilitating the breakdown of H2O2 to H2O and O2 gas.
Procedure:
A. Catalase
Preparation of catalase enzyme.
a. Wash, peel and grate a potato.
b. Place the grated potato in 100 ml distilled water.
c. Let stand for 10-15 minutes and stir occasionally.
d. Strain through a cheese cloth and squeeze to get as much extract as possible.
e. Filter the extract using a filter paper and use beaker as a receiving vessel.
f. Cover the beaker
g. Use the extract for the subsequent tests.

1. Test for the presence of proteins

a. Perform Biuret test on 2 ml of the extract.
b. Observe and note for the results

2. Test for catalase activity

a. Add one drop of 3% H2O2 to 1 ml of the potato extract.
 b. Observe and note for the reaction.
c. Add 1 ml of Benzidine solution.
d. Note for the formation of blue to green coloration.

3. Test for oxidase activity in milk

a. Place 5 ml of milk in each of the 3 test tubes.
b. Label the test tubes 1, 2, and 3.
c. Heat tube 1 in a boiling water bath for 1 minute.
d. Add 1 ml of methylene blue to each test tube.
e. Add 1 ml of neutral formalin into test tube no. 1 and test tube no. 2.
f. Add 1 ml of water to test tube no. 3.
g. Add 1 ml of mineral oil to all the test tubes.
h. Observe and note for the reaction.
i. Place all 3 test tubes in a boiling water bath.
j. Note the time it takes for each test tube to have complete decolorization.

B. Amylase
Preparation of Salivary Amylase
a. Rinse your mouth several times with water.
b. Collect 1 ml of saliva.
c. Dilute the saliva by adding 7 ml of distilled water.
d. Enzyme specificity
1. Place 2 ml of 0.2 M phosphate buffer (pH = 6.7) and 1 ml of 0.9% NaCl solution in a test tube.
2. Add 2 ml of cooked starch solution and 1 ml of salivary amylase.
3. Set aside and allow to stand for 15 minutes at room temperature.
4. Stir the mixture and place 1 drop in a spot plate.
5. Add 1 drop of iodine solution.
6. Repeat procedures d and e at 10 minutes intervals for 1 hour in separate spots.
7. Record your observations.

EXPERIMENT 6

Introduction:
Several factors affect enzyme activity. These factors include substrate concentration, enzyme
concentration, temperature, pH of the reaction and the presence of activators or inhibitors.
As the temperature rises, reacting molecules have more and more kinetic energy. This increases the
chances of a successful collision and so the rate increases. This optimal temperature is usually around human
body temperature (37.5 °C) for the enzymes in human cells. Above this temperature the enzyme structure
begins to break down (denature) since at higher temperatures intra- and intermolecular bonds are broken as
the enzyme molecules gain even more kinetic energy.
 OPTIMUM TEMPERATURE (Body) = 37-40°C
BELOW OPTIMUM TEMPERATURE = Enzyme action decreases
ABOVE OPTIMUM TEMPERATURE = Enzyme action increases (2x more);
60°C and above = DENATURATION due to the nature of enzymes

Each enzyme works within quite a small pH range. There is a pH at which its activity is greatest (the
optimal pH). This is because changes in pH can make and break intra- and intermolecular bonds, changing the
shape of the enzyme and, therefore, its effectiveness.

OPTIMUM pH = 7 (neutral)
ABOVE or BELOW = no activity
Examples: Pepsin = 1.6-1.8
Lactase = 5.7
Trypsin = 7.8

The rate of an enzyme-catalyzed reaction depends on the concentrations of enzyme and substrate. As
the concentration of either is increased, the rate of reaction increases. For a given enzyme concentration, the
rate of reaction increases with increasing substrate concentration up to a point, above which any further
increase in substrate concentration produces no significant change in reaction rate. This is because the active
sites of the enzyme molecules at any given moment are virtually saturated with substrate. The
enzyme/substrate complex has to dissociate before the active sites are free to accommodate more substrate.
Provided that the substrate concentration is high and that temperature and pH are kept constant, the rate of
reaction is proportional to the enzyme concentration.

Procedures:

A. Effect of Temperature
1. Place 5 ml of 1% cooked starch solution each in three test tubes.
2. Add 1 ml of saliva to each of the three test tubes.
3. Place the first test tube in a water bath with the temperature controlled at 40° C.
4. Place the second test tube in a water bath with the temperature controlled at 60° C.
5. Place the third test tube in a water bath with the temperature controlled at 10° C by addition of ice
cubes.
6. Take a drop of the reaction mixture from test tube 1 and test with iodine solution on a spot plate every
5 minutes for 30 minutes.
7. Do the same for the other two test tubes.
8. Tabulate your results.

B. Effect of pH
1. Prepare 4 test tubes containing equal amounts of egg white.
2. Place the test tubes in a boiling water bath to coagulate the egg white.
3. Cool and mark the height of the solidified egg white.
4. Add the following reagents to each test tube:
Test tube 1 – 3 ml of 2% pepsin + 6 drops of 0.4% HCl
 Test tube 2 – 3 ml of 2% pepsin + 6 drops of 0.4% Na2CO3
Test tube 3 - 3 ml of 2% pancreatin + 6 drops of 0.4% HCl
Test tube 4 – 3 ml of 2% pancreatin + 6 drops of 0.4% Na2CO3
5. Determine the extent of digestion by measuring the amount of egg white dissolved.
6. Perform Biuret test on 1 ml of supernatant liquid from each test tube and 0.5% peptone standard.
7. Compare the result of the Biuret test of the supernatant liquid to 0.5% peptone standard.

C. Influence of Co-enzymes
1. Mix 0.5 g yeast and 0.5 g of white sand in a pestle.
2. Grind thoroughly using a mortar.
3. Add 10 ml of distilled water.
4. Transfer the mixtures in a tube.
5. Centrifuge for 10 minutes at 100 rev/minute.
6. Separate the supernatant liquid from the residue carefully using a pipette so that the residue is not
disturbed.
7. Prepare 4 test tubes as follows:
Test tube 1 – 2 ml of sucrose solution + 5 ml distilled water
Test tube 2 – 2 ml of sucrose solution + 5 ml supernatant liquid
Test tube 3 – 2 ml of sucrose solution + 5 ml distilled water + pinch of residue
Test tube 1 – 2 ml of sucrose solution + 5 ml supernatant liquid + pinch of residue
8. Mix the contents in the test tubes thoroughly.
9. Place the test tubes in a water bath with temperature controlled at 40° C for 1 hour.
10. Add 5 ml of Benedict’s reagent to each of the test tubes and place in a boiling water bath.
11. Observe for the formation of a yellow or brick red precipitate.

EXPERIMENT 7
Introduction:
Nucleic acids are polymers of nucleotide. There are two types of polymers namely DNA or
deoxyribonucleic acid and RNA or ribonucleic acid. DNA and RNA are differentiated by their sugar components,
deoxyribose for DNA and ribose for RNA. The bases are the same except that uracil replaces thymine in RNA.
Nucleic acids are important because they are closely associated with chromosomes responsible for the storage
and transport of genetic materials among others.DNA is present only in the nucleus of the cell while RNA in the
cytoplasm although a few are found in the nucleus.
Procedures:
1. Prepare 10 ml of 1% NaOH and add 50 ml of water.
2. Add 20 g dry yeast.
3. Heat on a water bath of ½ hours with occasional stirring.
4. Remove from heat and filter at once.
5. Allow the filtrate to cool then faintly acidify with acetic acid (use litmus paper).
6. Filter again and heat to evaporate solution until it reaches 20 ml in volume.
7. Allow cooling to 40°C.
8. To 36 ml of 95% alcohol, add 4 ml of concentrated HCl.
9. Pour the yeast solution with vigorous stirring into 40 ml of 95% alcohol solution.
10. Allow to settle and decant.
11. Wash the residue twice with 95% alcohol and twice with ether.
12. Transfer the residue to a filter paper and air dry.
13. Perform the following tests on the residue

A. Solubility test
1. Test the solubility of a pinch of RNA in the following reagents:
a) cold water
b) hot water
c) alcohol
d) dil. HCl
e) dil. NaOH

2. Test for the Components of RNA: Boil a small amount of RNA in 10 ml of 5% H2SO4 for 10 minutes.
Perform the following tests on the solution.
a. Test for Sugars (Molisch test)
1. Add 2 drops of Molisch reagent and mix thoroughly.
2. Incline the test tube and carefully pour down the sides of the test tube 5 ml of conc. H 2SO4.
3. Note the color at the junction.

b. Test for proteins

1. To 3 ml of the solution, add 5 ml of 10% NaOH and 1% CuSO4.
2. Note the color produced.

c. Test for Phosphates

1. To 3 ml of the sample, add a slight excess of NH4OH.
2. Acidify with dil. HNO3.
3. Add 5 drops of ammonium molybdate and warm.
4. Note the color of the precipitate.

d. Test for purines

1. To 3 ml of the sample, add an excess of NH4OH and a few drop of AgNO3.
2. Note the color of the reaction.