BIOCHEMISTRY LABORATORY

LABORATORY EXPERIMENTS 1TO 8

Outline • Physiologic
I. pH and Buffers
buffers:
II. Carbohydrates o Bicarbonate
III. Lipids o Orthophosphat
IV. Proteins o Proteins
V. Enzymes
VI. Nucleic Acids C. PH DETERMINATION
VII. Principles of Chromatography
VIII. Nutrition 1. Prepare the following samples:
a. Defibrinated blood
I. PH AND BUFFERS b. fresh milk
A. pH c. Freshly voided urine
2. Determine the approximate pH of the sample
• Defined as the negative log of the hydrogen using the pH paper by following these steps:
ion concentration a. Dip a piece of pH paper in the sample for
about 10 seconds. Remove the paper and
pH = - log [H+]
place it on a watch glass.
• pH of a solution is the common logarithm b. Match the color produced in the pH paper
of the reciprocal of the hydrogen ion with the color chart to determine the pH of
concentration as expressed as: the sample.
c. Record the results.
pH = log 1/ H +

• Detected and measured by using a pH meter

• Most common detectors are the color changes
of acid-base indicators
• An acid-base indicator is a weak acid
• The ionized and unionized forms of the indicator Figure 1. pH color scale
have different colors
• When: 3. Determine the approximate pH of the sample
o H+ = OH- →NEUTRAL solution using the pH meter.
o H+ > OH- →ACIDIC solution
o H+ < OH- →BASIC solution D. RESULTS
HIn + H20 ↔ H+ + In
Acid color Basic color
• When indicator is placed in an acid solution, the
equilibrium is shifted to the left and the Hln form
predominates
• In an alkaline solution, the ln- form predominates
• The color of the solution depends upon the ratio
of Hln to ln and is related to pH in the following
equation:
Log10 [In] = pH – pKa

B. BUFFERS

• a buffer solution is one that resists a change in Figure 2. Experiment Results based on the pH scale
pH when a small amount of acid or base is
added E. QUESTIONS
• contains a weak Bronsted acid, HA and its
conjugate base, A−. 1. What is the H+ concentration of a phosphate
o i.e. mixture of acetic acid and sodium acetate buffer solution if its pH is 7.4?
• o pH = log 1/ H+

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o H+ = 10 -7.4
o H+ = 3.98 x 10-8 mol/L 5. What is the normal pH of blood? Urine?
2. What is the pH of a 0.125 M NaOH solution? Gastric juice? Why is it necessary to know
(Clue: pH + OH = 14) the pH of these physiologic fluids?
o pOH = log 1/ H+ o Normal pH:
o pOH = log 1/ 0.125 M = 7.35 –
o pOH = 0.903  Blood 7.45
o pH + pOH = 14  Urine = 4.8 – 8.0
 Gastric juice = 1.5 – 3.5
o pH= 14 – 0.903
o pH = 13.097 o It is essential to be familiar of the pH
values of these physiologic fluids in order
3. What is the significance of buffer systems in to be aware if the body is working
humans? optimally. Deviations from these normal
o Regulation of body fluid pH is one of the most pH values can cause severe health
important physiological functions of problems and even death.
homeostasis, because activity of most chemical
reactions via enzyme proteins is dependent on 6. What is the biochemical relevance of pH?
fluid pH. Measurement of pH is one of the most
•
o To maintain homeostasis of body fluid pH, important and frequently used
various buffering systems are utilized in addition procedures in Biochemistry. The pH
to proton excretion from the cytosol to the affects the structure and activity of
extracellular space and ultimately outside of the biological macromolecules; for example,
body. the catalytic activity of enzymes.
o However, if production of organic acid is Measurements of the pH of the blood
elevated or the buffering and excretion systems and urine are commonly used in
are impaired, body fluid turns acidic, leading to diagnosing disease.
abnormal conditions. 7. Calculate the pH of a 0.01M HCl.
o Ex.: When carbon dioxide dissolves in blood, it o pOH = log 1/H+
decreases the pH value, thereby increasing the o pOH = log 1/0.01
acidic content of blood. In this case, alkaline o pOH = 2
buffers come into play. They tend to mix with the
plasma of blood and then neutralize its value. II. CARBOHYDRATES
The same happens in the plasma when the
alkaline value of blood increases. In this case, • Compounds that contain
acidic buffers in the blood plasma play their role. carbon, ketones, or
If the alkalinity or the acidity of blood pertains for substances that can be
a longer period, the body gets into a hazardous hydrolyzed into these
state, which if left unaddressed, can prove fatal. hydroxylated aldehydes or
4. What is the Henderson-Hasselbalch equation? ketones.
Explain. • Medically important carbohydrates
which contains 6 carbons
pH = pKa + log [A-]/[HA] (hexoses):
o Glucose
o Where:  most frequently assayed
 pKa = negative log of Ka carbohydrate because it is
 Ka = equilibrium constant for readily metabolized by
dissociation of weak acid; describes most tissues
tendency of HA to donate H+  glucose levels may be
o The Henderson-Hasselbalch equation determined during the fasting
provides a general solution to the quantitative
state or periods of stimulation
treatment of acid-base equilibrium in biological
when the body is given an
system. It describes relationship between pH
oral glucose load.
of a solution, Ka of acid and extent of its
 TESTS – postprandial blood
dissociation.

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sugar (PPBS) , oral glucose color at the junction of the two

tolerance test (OGTT) liquids.
o Fructose • Principle:
o Galactose o Carbohydrates when treated with
• Disaccharides – carbohydrates that concentrated sulfuric acid undergo
contain 2 sugar moieties dehydration to give furfural
o Lactose (glucose + galactose) derivatives. These compounds
o Sucrose (glucose + fructose) condense with alpha naphthol to form
o Maltose (glucose + glucose) colored products. Pentoses yield
furfural while hexoses yield 5-hydroxy
methyl furfurals.
A. GLUCOSE DETERMINATION • Theoretical Result:
o Appearance of a purple ring at the
1. Chemical Method
junction of two liquids
• Depends upon the reducing
properties of this sugar.
Because of its lack of
specificity, this method is no
longer used clinically.
2. Enzymatic Method
• Determine glucose levels
yield maximum specificity
for glucose estimations.
• Glucose can be measured
using the reaction catalyzed
by the enzyme, glucose
oxidase.
Figure 3. Molisch’s Test Result, Presence of Purple Ring.
B. REDUCING PROPERTY OF CARBOHYDRATES
2. Fehling’s Test
1. Objective • Procedure:
• The students should be able to o Add 4ml of dist. Water to 1ml of
demonstrate how carbohydrates Fehling’s solution. Place 1ml of the
reduce Fehling’s or Benedict’s diluted solution in separate test
reagents. tubes. Heat the tubes in a water bath
2. Fehling’s or Benedict’s Test for 1 minute then add 8-10 drops of
a. Heat to gently boiling each sugar solution (glucose,
2ml of fehling’s or lactose, sucrose, starch and maltose)
benedict’s solution to the heated Fehling’s solution until
color reaction takes place and
b. Then add 2-5 drops of the continue boiling for another 2
carbohydrate solution minutes.
c. Continue the time required to show • Principle
any evidence of reduction.
o The presence of aldehydes
C. QUALITATIVE TESTS FOR CARBOHYDRATES but not ketones is detected by
1. Molisch’s Test reduction of the deep blue solution
• Procedure: of copper (II) to a red precipitate of
o To a 1ml of 1% glucose solution in insoluble copper oxide. The test is
the test tubes, add 2 drops of commonly used for reducing sugars
Molisch’s reagent and mix but is known to be NOT specific for
thoroughly. Incline the tube and allow aldehydes. Aldehydes are oxidized,
1ml of conc. H2SO4 to flow down the giving a positive result, but
side of the tube so that layers are ketonesdo not react, unless they
formed. Note the purple or violet are alpha-hydroxy- ketones. The

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bistartratocuprate(II) complex o When Benedict’s solution and

oxidizes the aldehyde to a
carboxylateanion, and in the
process the copper(II) ions of the
complex are reduced to copper(I)
ions. Red copper(I) oxide then
precipitates out of the reaction
mixture, which indicates a positive
result.
• Theoretical Result:
o Positive result is detected by
formation of a reddish
precipitate
o Glucose, lactose, and
maltose – gives a positive
result
o Sucrose
 Does not react with Fehling’s
reagent
 It is a non-reducing sugar
because the anomeric carbon
of glucose in involved in the
glucose-fructose bond, hence
it is not free to form the
aldehyde in the solution
o Starch
 Also a non-reducing sugar,
thus it will not react with
Fehling’s reagent
simple carbohydrates are heated,
the solution changes to orange
red/ brick red. This reaction is
caused by the reducing property of
simple carbohydrates.
o The copper (II) ions in the
Benedict’s solution are reduced to
Copper (I) ions, which causes the
color change. The red copper(I)
oxide formed is insoluble in water
and is precipitated out of solution.
This accounts for the precipitate
formed.
o As the concentration of reducing
sugar increases, the nearer the final
colour is to brick-red and the greater
the precipitate formed. Sometimes a
brick red solid, copper oxide,
precipitates out of the solution and
collects at the bottom of the test tube.
• Actual Result:
o Glucose, Lactose, Sucrose –
positive result
o Starch, Maltose – no change in color
• Theoretical Result:
o Positive Result – Formation of a
reddish precipitate means that the
sugar in the solution is reducible
o Negative Result - No change in
color (remains blue), the sugar in
the solution is non-reducible
o Glucose, lactose, and maltose –
should give a positive result
o Starch and Sucrose – should give a
negative result, since starch and
sucrose are non-reducing sugars

Figure 4. Fehling’s Test. (from left to right: glucose, lactose,

sucrose, starch, and maltose).

3. Benedict’s Test
• Procedure:
o 1ml of Benedict’s reagent, add 10
drops each of 1% sugar solution in
separate test tubes. Boil in a water
bath for two minutes. Allow to cool
and observe the color changes and
formation of precipitate
• Principle: Figure 5. Benedict’s Test Result.

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4. Barfoed’s Test
• Procedure:
o Mix 1ml of Barfoed’s reagent with 10
drops each of the 1% sugar
solutions. Heat in a boiling water
bath for 5mins then allow to cool &
stand for 15mins. Note for the
presence of a small amount of brick-
red precipitate
• Principle:
o When Barfoed’s reagent mixes with
series of monosaccharide or
disaccharide and warmed in bubbling
water shower, they react and a
precious stone solution is formed.
Copper acetic acid derivation which is
available in Barfoed’s reagent of
copper oxide and gives block red
solution when it reacts with
monosaccharide or disaccharides.
Monosaccharide responds quickly
while disaccharide responds gradually
• Actual Result:
o Glucose and Sucrose – a small
amount of brick red precipitate is
present
o Starch – no change in color
• Theoretical Result:
o Positive result – formation of a
brick red color; it implies that
monosaccharides are present
in the solution
o Glucose and Sucrose –
Should give positive result
since they are
monosaccharides
o Starch – A polysaccharide,
hence should give a negative
result
Figure 6. Barfoed’s Test. Left: Glucose and Sucrose, Right: Starch.
5. Picric Acid Test
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LABORATORY EXPERIMENTS 1 AND 2
• Procedure:
o Add 10 drops of saturated picric
acid solution and 4 drops of 10%
Na2CO3 solution to 1ml each 1%
glucose, lactose, and maltose
solutions in separate test tubes.
Warm and note the formation of
yellow to red brown precipitate
• Principle:
o It is another test for the detection
of reducing sugars. The reducing
sugars react with Picric Acid
(yellow) to form a red coloured
Picramic or dark brown-red.
• Actual Results
o Lactose, Maltose, and Glucose
– changed color; positive
• Theoretical Result:
o Positive Result – Formation of
red to dark brown-red
precipitate
o Lactose, Maltose, and Glucose –
should give a positive result
Figure 7. Picric Acid Test. (from left to right: lactose maltose and
glucose).
6. Tollen’s Test
• Procedure:
o To 1ml of 1% glucose solution, add
few drops of Tollen’s reagent. Warm
in a water bath for several minutes
and observe the results
• Principle:
o Aldehydes are oxidized to carboxylic
acid, and silver(I) is reduced to silver
metal, which deposits as a think film
on the inner surface of the glass.
• Theoretical Result:
o Positive Result – formation of a sliver
mirror
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Figure 8. Tollen’s Test.
[HINDI NATIN GINAWA YUNG EXPERIMENT NA ITO
SO THIS IS JUST COPIED FOR EXAM PURPOSE
ONLY KASI BAKA KASAMA]
D. QUANTITATIVE TESTS FOR CARBOHYDRATES
• Preparation of protein-free blood filtrates
o The presence of proteins
interferes with many chemical
determinations used in the analysis
of blood constituents, primarily the
analysis for compounds containing
amino acids or amino nitrogen, and
those involving reduction or
oxidation of metal ions.
o Some procedures require that
proteins be removed from a sample
and the analysis be made on protein
free blood filtrates.
1. Folin-Wu Method
• One of the most commonly used methods
for the preparation of a protein free
filtrate. It involves removal of the protein
by precipitation with tungstic acid and
subsequent filtration.
• Reagents: Sodium tungstate, 10% and
Sulfuric acid, 0.67 N
• Procedure:
o Into a 50ml Erlenmeyer flask place
accurately measured quantities of 1.5
ml distilled water, 0.5 ml of blood,
0.5ml of 10% sodium tungstate,
0.5ml of 0.67 N sulfuric acid
o After the addition of each
component and before the addition
of the next, mix well.
o After the addition of sulfuric acid,
stopper the flask and shake by
rotation.
o Let it stand for 10 minutes
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LABORATORY EXPERIMENTS 1 AND 2
o The color of the mixture should turn
form red to dark brown
o IMPORTANT (should you need more
than the usual amount of sulfuric
acid, note the total volume of acid
used and correct for the dilution of
the blood)
o Removal of the precipitate can be
done by either centrifugation of
filtration as described
o Transfer mixture into a 15ml
centrifuge tube and spin at 2000 rpm
for 20 minutes. Save clear supernate
o Filter the mixture into a clean dry
flask, using a folded filter paper cut
just enough to hold it. If there is a
slight turbidity at the beginning, pour
the first few drops of filtrate back on
the filter. This filtrate should be water
clear.
o In general, 0.5ml of blood will yield
about 3ml of filtrate.
2. Nelson-Somogyi Method
• Reagents: Zinc sulphate, 10%, ans NaOH,
0.5N
• Procedure:
o withdraw 0.02 ml form a finger prick
with a Sahli pipette.
o Expel the blood into a micro
test tube containing 0.8ml
distilled water.
o Rinse the pipette by drawing the
water and then expelling the rinsing
back into the tube
o Withdraw another 0.02ml of blood
using the same Sahli pipette and
expel into the same test tube.
o Treat hemolyzed blood with 0.1ml of
10% zinc sulphate and 0.1ml of 0.5 N
NaOH solns
to precipitate the proteins and the
non-sugar reducing substances.
o Shake the tube sideways and
centrifuge at 1500 rpm for 5
minutes.
o Pipette out into another test tube.
3. Modified Determination of Sugar
• The level of glucose in the blood reflects a
dynamic state between the production by
the liver and its utilization by other tissues.
It is important that the blood glucose level
be maintained within
o Cool test tube in tap water
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for another 10 mins.
o Add 0.5ml of
phosphomolybdic acid
color reagent
o Add 3.5 ml distilled water to
all tubes. Mix thoroughly and
read the absorbance against
the blank at 420nm.
B. Questions
certain limits as the brain depends heavily
on
• The methods for measurement of glucose
can be classified into three general types
o Oxidation-reduction
o Condensation
o Enzymatic reactions
• The following procedure is an oxidation-
reduction method wherein the blood filtrate
prepared by the Nelson-Somogyi method
is heated with the alkaline copper reagent
of Folin-Wu.
• The cuprous oxide formed is treated
with phosphomolybdic acid reagent
resulting in the formation of a blue color
that is measured
spectrophotometrically.
• Reagents
o Alkaline copper solution –
Reagent A: Dissolve 5.0g CuSO4 in
a volumetric flask with distilled water
to make 100ml
 Reagent B: Weigh the ff 3.5g
Na2CO3 (anhydrous) and 1.10g
NA2C4H4O6 (sodium tartrat).
Dissolve in distilled water to make
100ml (On the day of experiment,
mix 1 part of reagent A with 9
parts of reagent B.)
o Phosphomolybdic Acid Reagent
 Na2MOO4 C.P- weigh 3.0g.
Add distilled water to make
100ml.
 Conc. H2SO4 (sp gr1.84) –
Pipette 13.5ml and add about
50ml distilled water. Cool and add
more distilled water to make
100ml. Put 50ml of A. in a 100ml
volumetric flask and add 22,5ml of
85% H3PO4 and 15ml of B. blow
air through this soln. to remove the
bromine and add 7.5ml glacial
acetic acid. Make up to the final
volume of 100ml distilled water.
• Procedure:
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LABORATORY EXPERIMENTS 1 AND 2
o Place 0.5ml of protein-free filtrate
(prepared by the Nelson-Somogyi
method) into a testtube.
o Into another test tube 0.5ml each
of the standard glucose soln.
o Prepare a blank tube containing
0.5ml of distilled water.
o Add 0.5ml of alkaline copper
reagent to all tubes.
o Cover tubes with aluminum foil
glucose as a source of energy.
and heat them in boiling water
bath for 10 mins.
Qualitative Test for Carbohydrates
1. Aside from the reducing tests,
the presence of carbohydrates
may be found using other tests.
Described the Molisch test and
how it detects the presence of
carbohydrates in a mixture?
• The Molisch test is a general
test for the presence of
carbohydrates. This test is
useful for identifying any
compound which can be
dehydrated in the presence
H2SO4. Carbohydrates when
treated with concentrated
sulfuric acid undergo
dehydration to give furfural
derivatives. These compounds
condense with alpha naphthol
to form colored products.
Pentoses yield furfural while
hexoses yield 5- hydroxy
methyl furfurals.
2. Enumerate some other tests that
use the reducing properties of
sugars.
• Fehling’s Test –
carbohydrates with a free or
potentially free carbonyl group
have the ability to reduce
solution of various metallic ions
such as Fehling’s solution
(cuprous oxide)
• Benedicts Test – Detects
reducing sugar when a red
brown copper sulfide
precipitate is seen
• Picric Acid Test –
Reduction of picric acid
(yellow) to picramic acid
(mahogany red) indicates
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presence of reducing
sugars
3. What could be a practical
use for these tests,
considering their lack of
specificity?
• These tests, such as
Benedict’s or Fehling’s test,
can determine whether sugars
are present in the urine, which
may indicate Diabetes Mellitus
Quantitative Test for Carbohydrates
1. How are proteins removed in
biological samples to produce
protein free filtrate blood for
chemical analysis? Give the
principle involved in the
methods given.
a. FOLIN-WU METHOD
• Principle: Protein is
removed from the
sample by precipitation
with tungstic acid and
centrifugation or
filtration. Sugar present
in the filtrate reduces
alkaline copper tartrate.
The cuprous oxide
produced in this
reaction is then treated
with phosphomolybdic
acid to produce a blue
compound. The color
intensity is directly
proportional to the
glucose concentration.
b. SOMOGYI-NELSON METHOD
• Principle: The reducing sugars when
heated with alkaline copper tartrate
reduce the copper from the cupric to
cuprous state and thus, cuprous oxide
is formed. When cuprous oxide is
treated with arsenomolybdic acid, the
reduction of molybdic acid to
molybdenum blue takes place. The
blue color developed is compared
with a set of standards in a
colorimeter at 620 nm.
2. What is the basis of blood samples
determination in this laboratory exercise?
• Blood samples must be free of
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LABORATORY EXPERIMENTS 1 AND 2
proteins because these
macromolecules interfere with
chemical determinations used in the
analysis of blood constituents,
primarily the analysis for compounds
containing amino acids or amino
nitrogen, and those involving reduction
and oxidation of metal ions.
3. Diagnosis of diabetes mellitus may be
made by measurement of plasma
glucose in the fasting state (12-14 hours
fast). Why?
• When fasting the hormone glucagon is
stimulated and this increase plasma
glucose levels in the body. If a patient
doesn’t have diabetes, their body will
produce insulin to rebalance the
increased glucose levels. However,
people with diabetes either don’t produce
enough insulin to rebalance their blood
sugar or their body is not able to use the
insulin effectively enough. Also, when
blood glucose levels are tested, people
with diabetes will have blood sugar levels
significantly higher than people who do
not have diabetes.
4. Give the normal values for blood
glucose using different methods
commonly used in the diagnostic
laboratory.
• Glucose tolerance test - lower than 140
mg/dL
Random glucose test - 79–160 mg/dl
III. LIPIDS
• Constitute a large heterogenous group of
unrelated physiological and chemical
substances classified together because they are
fat-like substances
• Insoluble in water but are soluble in organic
solvents such as chloroform, ether, carbon
tetrachloride, alcohol and benzene
• Important lipids include:
o Triacylglycerols
o Cholesterol
o Derivatives of arachidonic acid
• Rich energy source
• Play an important role as structural components
in practically all plant and animal cells
• In human body, lipids are found mostly in
cellular structures like the cell membrane, in vital
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 LABORATORY EXPERIMENTS 1 AND 2

organs like brain and various tissues like
nervous tissue.
A. PROCEDURE
1. SOLUBILITY TESTS
a. Pipette 1 ml of the following solvents in
separate stoppered vials or test tubes:
distilled water, ethyl alcohol, ether,
chloroform, benzene, 5% HCl, 5%
NaOH, acetone.
b. From a pipet or a dropper, add 1-2
drops of cottonseed oil in each vial and
shake thoroughly. Record the time
required for the oil dissolve.
b. Test the reaction of fresh coconut oil
with phenolphthalein, methyl orange and
pH paper.
c. Do the same reaction tests with the
rancid coconut oil.
5. LIEBERMANN-BURCHARD TEST FOR
CHOLESTEROL
a. Place a few crystals of cholesterol in a
dry evaporating dish. Add 2 ml of
chloroform and 10 drops of acetic
anhydride. Mix thoroughly.
b. Add 2-3 drops of concentrated H2SO4
and shake. Note the color changes
during the first few minutes.

c. On different spots of a piece of coupon B. RESULTS

bond paper, place 3 drops of each of
these mixtures: 1. SOLUBULITY TESTS
• Lipids are non-polar organic
• cottonseed-ethyl alcohol
compounds. The physical properties of
• Cottonseed-ether fatty acids and of compounds that
contain them are largely determined by
d. Allow the solvents to evaporate and the length and degree of unsaturation of
compare the solubility of the oil in two the hydrocarbon chain. The non-polar
solvents. hydrocarbon chain accounts for the poor
solubility of fatty acids in water.
2. TEST FOR UNSATURATION OF FATTY Solubility of a substance depends on a
ACIDS simple rule of thumb “like dissolves like”
a. To 6 drops of CCl4add 3 drops of oleic which indicates that a solute will
acid. Then add bromine water in dissolve best in a solvent that has a
CCl4drop by drop into the mixture, similar chemical structure to itself.
shaking the vial after each addition. Solubility depends primarily on its
Note the number of drops needed to polarity.
produce a faint orange color.
TABLE 1. SOLUBILITY OF LIPID IN DIFFERENT SOLVENTS
b. Into each of 3 test tubes or stoppered
vials, place 2 ml of CHCl3. Add to each Solvent Oil Time Required
test tube 0.2 g of palmitic acid, 4 drops for Oil to
oleic acid and 4 drops of cottonseed oil Dissolve
respectively. Shake each tube 5% NaOH 1-2 drops of Did not dissolve
thoroughly to dissolve the contents. cottonseed oil
3. ACROLEIN TEST 5% HCl 1-2 drops of Did not dissolve
a. Prepare 2 test tubes. Place in: cottonseed oil
• Test tube No. 1 – 2 drops of Ethyl alcohol 1-2 drops of Did not dissolve
glycerol + a pinch of KHSO4 cottonseed oil
• Test tube No. 2 – 2 drops of Distilled water 1-2 drops of Did not dissolve
cottonseed oil + pinch of cottonseed oil
KHSO4
b. Heat each tube over a low flame. Note Ether 1-2 drops of 2 seconds
the odor produced. cottonseed oil
Benzene 1-2 drops of 3 seconds
4. TEST FOR RANCIDITY cottonseed oil
a. Prepare 6 tubes or vials. In each of the
Acetone 1-2 drops of 2 seconds
tube nos. 1, 2 and 3 places 5 drops of
cottonseed oil
fresh coconut oil, and in each of the test
tubes nos. 4,5 and 6 place 5 drops of Chloroform 1-2 drops of 3 seconds
rancid coconut oil. cottonseed oil

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 LABORATORY EXPERIMENTS 1 AND 2

Figure 12. Test for Unsaturated Fatty Acids II. From L to R:

Palmitic acid, oleic acid, cottonseed oil

TABLE 3. RESULT OF TEST FOR UNSATURATED

FATTY ACIDS
Mixture Result (color)
Figure 9. SolubilityTest.(A) from left to right: NaOH, 5% HCl, Ethyl 2 ml of CHCl3 + 0.2 g of Clear
Alcohol and Distilled Water (B) From left to Right: Ether, Benzene, palmitic acid
Acetone and Chloroform
2 ml of CHCl3 + 4 drops Clear
of oleic acid
TABLE 2. EVAPORATION AND SOLUBILITY OF OIL 2 ml of CHCl3 + 4 drops Turbid
Mixture Evaporation of cottonseed oil

Cottonseed-ethyl alcohol Faster 3. ACROLEIN TEST

Cottonseed- ether Slower • A test for the presence of glycerol
and thus the presence of fats and
oils.
• Positive result: A pungent irritating
odour of acrolein confirms the
presence of oil or fat.
TABLE 4. RESULT OF ACROLEIN TEST
Test tube No. Odor
1 No Odor
Figure 10. Evaporation of Cottonseed. (Left)Ethyl alcohol (Right) 2 Oily Smell
Ether (See Table 2 above for evaporation rate)

2. TEST FOR UNSATURATION OF FATTY 4. TEST FOR RANCIDITY

ACIDS • Rancidity testing determines the
• This test is specific for the double level of oxidation in a sample. When
bonds in the lipid’s chain. lipids (fats and oils) go rancid, its
a. No change in color after 106 drops. nutritional value is compromised,
and the lipids will take on a rancid
taste and odor. Hydrolysis of the
fats to give the fatty acids and
glycerol and oxidation of
unsaturated parts of the fatty acids,
yielding volatile acids, aldehydes
and ketones which have a
disagreed odor are catalyzed by
microorganisms from the air.
Figure 11. Test for Unsaturation of Fatty Acids. No change of
color even after 35 drops.

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 LABORATORY EXPERIMENTS 1 AND 2

TABLE 5. TEST FOR RANCIDITY RESULTS hydrophobic than the carboxyl group
which is soluble in water.
Sample Reagent Added Result
2. Explain why cis-form is the predominant
configuration of unsaturated fatty acids.
Fresh Coconut Phenolphthalein Colorless, No - Cis-form configuration is more
Oil Reaction predominant in unsaturated fatty acids
Fresh Coconut Methyl Orange Orange Color, since most of the fatty acids are in liquid
Oil form. The cis form of unsaturated fatty
acids is more fluid at biological
Fresh Coconut pH Paper Blue Lithmus temperatures and are more abundant in
Oil Paper to Red living organisms.
Rancid Phenolphthalein No Reaction
Coconut Oil 3. Why is the acrolein test a general test for
fats?
Rancid Methyl Orange Orange Color - Acrolein test is a general test for fats
Coconut Oil because it is used to detect the
Rancid pH Paper Blue Lithmus presence of glycerol or fat. When fat is
Coconut Oil Paper to Red treated strongly in the presence of a
dehydrating agent like potassium
bisulphate (KHSO4), the glycerol portion
of the molecule is dehydrated to form an
unsaturated aldehyde, acrolein that has
a pungent irritating odor.

4. What type of rancidity occurs in

Figure 13. Test for Rancidity.
5. LIEBERMANN-BURCHARD TEST FOR
CHOLESTEROL
• Also known as acetic anhydride
testis used for the detection of
cholesterol. The formation of a
green or green-blue color after a few
minutes means positive result.
Figure 13. Result: No change in color
C. QUESTIONS
1. Why are fatty acids insoluble in water?
- Carboxyl end of fatty acid is highly polar
while hydrocarbon chain of the fatty acid
is highly non polar. Fatty acids are
insoluble in water because there are
more hydrocarbons which are more
vegetable shortenings and how can it be
prevented?
- The type of rancidity that occurs in
vegetable shortenings is the oxidation
rancidity. The oxygen molecules interact
with molecules of the oil which causes
damage or changes to substance.
- This can be prevented by storing
vegetable shortenings in a dark, cool
place where it is less exposed to
oxygen.
5. Explain the cleansing action of
detergents and soaps.
- The cleansing action of soap is
determined by its amphipathic
properties- polar and non-polar
structures, as well as its solubility
characteristics. The long hydrocarbon
chain is non-polar and hydrophobic
(repelled by water); and the "salt" end of
the soap molecule is ionic and
hydrophilic (water soluble). Since the
dirt is mostly oily in nature which does
not dissolve in water, the carbon chain
dissolves in oily dirt and ionic end
dissolves in water. It leads to formation
of micelles. This forms emulsion in
water which is easily removed. In short,
oily dirt is dissolved in Carboxylic acid
part of soap and is removed.
6. Write the structure of the parent
compound of cholesterol.

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 LABORATORY EXPERIMENTS 1 AND 2

o Enzyme-linked immunosorbent assay

(ELISA) Western blot

MATERIALS
• Protein dispersion CuSO4
solution NaOH solution
Millon’s reagent Nitric acid
• Ammonium hydroxide
• Glacial acetic acid/glyoxylic acid solution Sulfuric
7. Explain the cooperative solvent effect of acid
lecithin and albumin. • Naphthol-alcoholic solution
- Lecithin and albumin are good • 0.1% Ninhydrin alcoholic solution Test
emulsifiers because they both have a
tubes, test tube holder, rack Bunsen
non-polar and a polar portion which
helps reduce the immiscibility of two burner
substances. When they work hand in • Beaker 25 ml
hand, they are able to hold polar
substances through their hydrophilic QUALITATIVE TESTS
molecules thus making the immiscibility 1. Biuret Reaction
of two substances possible. • Procedure
IV. PROTEINS a. 10 drops each of the following in separate
• Major constituents of every living cell test tubes: 1% albumin and
• Polymers of amino acids connected via peptide b. 1% glycine Add 5 drops of 10% NaOH and
linkages 3 drops of 1% CuSO4 to each test tube
c. Mix and observe formation of pink or violet
• Functions:
color
o Catalysts of biochemical reactions
o Regulate activities of various organs in d. Place a pinch of urea in dry test tube. Heat
body Counteract adverse effects of test tube over low flame until urea melts (do
antigens Transport molecular oxygen not char) and gas evolved.
o Serve as structural materials of
e. Cool, dissolve contents in 20 drops of
muscles, skin, hair, etc.
distilled water and add 5 drops of NaOH
o Make up majority of body’s
framework and substance and 3 drops of 1% CuSO4
• Determination of proteins is through colorimetric
assays • Results
o 1% albumin – formation of violet color
• Analysis of Nitrogen Content
o 1% glycine – formation of blue color
o e.g. Khendal method o Urea – formation of pink color
o Ultimate reference method for determining
protein concentration
• Methods for protein analysis
o Refractive index
o Turbimetric salting-out
procedures Colorimetry
o Chemical
precipitation Dye
binding
o Determination of mass values, refractive
index Turbidimetric salting-out
procedures Electrophoresis
o Immunoelectrophor Figure 16. Biuret Reaction
Esis Nephelometry

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 LABORATORY EXPERIMENTS 1 AND 2

2. Millon’s Reaction
• Procedure

a. Into 3 separate test tubes, place 20 drops

each of the ff: 1% albumin, 1% gelatin and
1% casein. Add 6 drops of Millon’s reagent
to each tube. Shake and boil in water bath
until appearance of red color.
b. Perform test on 20 drops of 1% phenol. Heat
in water bath if necessary. Compare the
result with (1)

• Result
o 1% gelatin – formation of pink color
o 1% casein – No reaction
o 1% albumin – formation of red color
o 1% phenol – formation of orange color Figure 18. Xanthoproteic Reaction. (1st tube: albumin | gelatin | 3rd:
phenol).

4. Ninhydrin Test
• Procedure

a. To 1 ml of neutral 0.2% albumin solution add

0.5 ml of 0.1% freshly prepared ninhydrin
solution. Cover test tube with marble and
boil over water bath. Note purple color
produced.
b. Repeat test with a) ammonia water, b)
0.2% urea, and c) 0.2% glycine instead of
0.2% albumin. Compare results.

• Result
Figure 17. Millon’s Reaction actual results. o 0.2% albumin – formation of blue color
o 0.2% urea – No reaction
o 0.2% glycine – formation of dark purple
3. Xanthoproteic Test color
• Procedure o Ammonia water – formation of red color
a. Into 3 separate test tubes, place 10 drops
each of the ff: 1% albumin and 1% gelatin.
Add 5 drops of concentrated HNO3 to each
test tube. Mix thoroughly. Note formation of
heavy white precipitate. Heat in water bath.
Observe change in color. Cool and add a
few drops of conc. NH4OH. Note change in
color intensity of substance.
b. Repeat test with 10 drops of 1% phenol
solution. Compare result with (1)

• Result

o 1% albumin – formation of yellow color Figure 19. Ninhydrin Reaction. (1st tube: 0.2% urea |
o 1% gelatin – No reaction 2nd: 0.2% albumin | 3rd: ammonia water | 4th: 0.2%
o 1% phenol – formation of red-orange color glycine).

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 LABORATORY EXPERIMENTS 1 AND 2

PROTEIN PRECIPITATION 3. Heat

• A factor which affects protein at varying degrees is
heat
• Heating an aqueous solution of protein may cause
precipitation
• Hydrogen bonds, ionic bonds and hydrophobic bonds
are destroyed because of increased molecular
vibration
• Protein undergoes intramolecular rearrangement,
rendering it insoluble but more readily digestible
This phenomenon is known as protein denaturation
METHODS (PROTEIN PRECIPITATION)
1. Concentrated Inorganic Acids
a. Place a pinch of powdered egg albumin in
each of 2 dry test tubes labeled 1 and 2. To
test tube 1, add 2 ml of distilled water then
place both in boiling water bath for 10
minutes with constant shaking. Remove test
tubes, cool to room temperature then add
2ml of distilled water to test tube 2. Filter
solutions and test both filtrates with Biuret
reagent – 5 drops
b. Perform Biuret test on 1ml of 1%
albumin solution. Compare result with
(1).

a. 3 test tubes prepared containing 1ml each of

1% filtered albumin solution
b. Add the ff reagents shaking carefully after
each addition
o Test tube 1 – concentrated HCl, 1ml
each
o Test tube 2 – concentrated H2SO4, 1ml
each
o Test tube 3 – concentrated HNO3, 1ml
each
c. Observe for precipitate formations then
add excess of reagent
d. Observe effects of excess reagent on
precipitate

2. Alcohol

a. Mix thoroughly and observe any formation of

precipitate. Test the solubility of this
precipitate in water.
b. Repeat test in 1% albumin solution using
70% alcohol instead of 95% alcohol.
Compare result with (1)
Figure 21. Heat, Protein Precipitation Test.
QUESTIONS
1. Enumerate some color-reaction tests for
proteins and their expected results.

Figure 20. Alcohol, Protein Precipitation Test.

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Table 6. Color of Reactions of Proteins.
2. State the principle involved in the different
color reactions for proteins.
a) Biuret Test
o Reacts with peptide bonds in proteins to
form a violet complex known as biuret
complex
o Two peptide bonds at least are required for
the formation of this complex
o Positive visible result: violet
Negative result: blue
b) Ninhydrin Test
o In the pH range of 4-8, all alpha amino acids
react with ninhydrin (triketohydrindene
hydrate), a powerful oxidizing agent to give
a purple colored product (dikeothydrin)
termed Rheumann’s purple
o All primary amines and ammonia react
similarly but without the liberation of
carbon dioxide
o The imino acids proline and hydroxyproline
also react with ninhydrin but they give a
yellow colored complex instead of a purple
one
o Besides amino acids, other complex
structures such as peptides, peptones and
proteins also react positively when
subjected to the ninhydrin reaction
c) Xanthoproteic Test
o Used to determine the presence of aromatic
amino acids such as phenyl alanine,
tyrosine, and tryptophan
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LABORATORY EXPERIMENTS 1 AND 2
o Aromatic groups in the amino acids will be
nitrated by HNO3. The nitro derivate show
an intensely yellow color. Because nearly all
proteins contain aromatics it is taken as a
protein-test either
o At alkaline pH, the color changes to orange
due to the ionization of the phenolic group
d) Millon’s Test
o Phenolic amino acids such as tyrosine and
its derivatives respond to this test
Compounds with a hydroxybenzene radical
react with Millon’s reagent to form a red
colored complex
o Millon’s reagent is a solution of mercuric
sulfate in sulfuric acid
e) Hopkin’s-Cole Test
o This test is specific for detecting tryptophan
The indole moiety of tryptophan reacts with
glyoxilic acid in the presence of
concentrated sulfuric acid to give a purple
colored product Glyoxilic acid is prepared
from glacial acetic acid by being exposed to
sunlight
f) Sakaguchi Test
o Under alkaline condition, alpha-naphthol (1-
hydroxynaphthalene) reacts with a mono-
substituted guanidine compound like
arginine, which upon treatment with
hypobromite or hypochlorite, produces a
characteristic red color
g) Reduced Sulfur Test
o Proteins containing sulfur give a black
deposit of lead sulfide when heated with
lead acetate in alkaline medium
h) Pauly’s Diazo Test
o Test is specific for the detection of
tryptophan or histidine
o Reagent used for this test contains
sulphanilic acid dissolved in hydrochloric
acid
o Sulphanilic acid upon diazotization in the
presence of sodium nitrite and hydrochloric
acid results in the formation of a diazonium
salt
o The diazonium salt formed couples with
either tyrosine or histidine in alkaline
medium to give a red colored chromogen
(azo dye)
i) Isatin Test
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o Imino acids such as proline and
hydroxyproline condense with isatin reagent
under alkaline condition to yield blue colored
adduct
j) Folin’s <cCarthy Sullivan Test
o Imino acids such as proline and
2. State and describe the different levels of
hydroxyproline condense with isatin reagent
under alkalin condition to yield blue colored
adduct
o Addition to sodium nitroprusside to an
alkaline solution of methionine followed by
the acidification of the reaction yields a red
color
o This reaction also forms the basis for the
quantitative determination of methionine
3. What is the practical use of these tests?
• Proteins are present in the living world and
different organisms irrespective of their size.
Since they form the structural and functional
basis of cells, it becomes necessary to
identify the presence of proteins.
Questions from Protein Precipitation
1. Differentiate denaturation from degradation
of proteins.
• Denaturation is the unfolding of proteins.
Certain chemicals may break up the internal
bonds that form the tertiary and secondary
structures while keeping the primary protein
structures intact. The primary protein
structure is formed by covalent bonds much
stronger than the non-covalent interactions
that form the secondary and tertiary
structures. When a protein denatures, it
changes from a three-dimensional shape to
its linear amino acid sequence. All biological
functions a protein had will be destroyed but
its primary structure remains intact.
• In contrast, when a protein degrades, it is
broken down on the primary level. This
means that the covalent bonds between
amino acids is broken. This can typically
happen after treatment with certain enzymes
called proteases. Degraded proteins can still
have a secondary or tertiary structure and
can even still have a biological activity.
However, in most cases, these can be
different from the original situation. Proteins
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LABORATORY EXPERIMENTS 1 AND 2
can be completely degraded to single amino
acids or very small peptide chains. Often
these small molecules are then recycled by
the body and included in newly synthesized
proteins
protein
a. Primary Structure
• Amino acids are in linear sequence, and
are joined by peptide bonds
b. Secondary Structure
• Secondary structure involves the folding
of the polypeptide chain into α-helices or
ß- pleated sheets due to formation of H-
bonds
• The functional groups of the polypeptide
chain have a tendency to form H-bonds
between them. Thus, when the reaction
of primary polypeptide chain is twisted
or coiled into a helix, hydrogen bonds
are formed between –CO and on –OH
group of amino acids facing each other
due to coiling of a polypeptide chain and
thus, a secondary structure of protein is
resulted.
c. Tertiary Structure
• Refer to the three-dimensional form of
the fully-folded polypeptide peptide
chain. The presence of covalent and
non-covalent bonds stabilizes the
structure
• In order to compress the very long spiral
chain in a globular form, these occur
folding- over and bending of the helices,
which result into tertiary structural
features. Tertiary structure permits
interaction over a vast surface and
creates interstices between the
polypeptide chains into which particular
reaction pattern can fit with optimal
accuracy so most tertiary proteins act
like catalysts. Enzymes have tertiary
structures.
d. Quaternary Structure
• Refers to the organization of
multiple polypeptides
• Structure formed by the non-covalent
interaction of two or more
macromolecules such as that formed by
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4 globin protein molecules to make
hemoglobin or that formed by histones
interacting with DNA to make a
nucleosome.
V. ENZYMES
A. CHEMICAL NATURE AND SPECIFITY
• An enzyme is usually a protein (it may be
nucleic acid in the case of peptidyl
transferase) synthesized in living cells,
which catalyzes thermodynamically possible
reactions, so that the rate of reaction is
compatible with the biochemical process
needed for the maintenance of the cell. In
short enzymes can be considered as
biochemical catalysts
B. PROCEDURE
• Preparation of Catalase
o Peel a potato and grate. Put it into 100
ml of distilled water. Let it stand for 10
min. Stir occasionally. Stain the mixture
C. RESULTS
through a cheese cloth and finally filter
and extract in the following tests.
1. Biuret Test
 To 2 ml of the extract mix 1ml of
10% NaOH solution. Mix thoroughly
and add 5 drops of 1% copper
sulfate solution
2. Test for Catalase Activity
 To 2 ml of the extract, mix 1 ml of
3% H2O2. Observe whether the gas
evolved supports combustion by
holding a glowing splinter over the
mouth of the test tube. Then add
1ml of 0.5% benzidine. Note the
formation of a blue green coloration
• Preparation of diute solution of Salivary
Amylase
o Rinse your mouth several times with
water. Then collect 1 ml of saliva.
Prepare 1:8 solution of the saliva by
adding 0.5 ml of saliva to 3.5 ml distilled
water. Use the prepared solution in the
following test.
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LABORATORY EXPERIMENTS 1 AND 2
• Test for Specificity of Enzyme Reaction
o Prepare 2 test tube, each containing 2
ml of 0.02 M phosphate buffer, pH 6.7
and 1 ml of 0.09% NaCl. To test tube
#1, add 1 ml cooked starch and 1 ml of
the solution of salivary amylase. To test
tube #2 add 1 ml of 0.1% glycogen
solution and 1 ml salivary amylase
solution. Let the test tube stand for 15
min. at room temperature.
o Stir the mixture in test tube #1 and
place 1 drop in an evaporating dish or
spot plate. Add 1 drop of iodine
solution. Observe the color produced.
Repeat this test at 5 min. intervals at 1
hour.
o Perform the test done in B in the
solution in test tube #2. Tabulate the
results.
• Preparation of Catalase
1. Biuret Test
 The copper atoms of Biuret
solution (CuSO4 and NaOH) will
react with several peptide bonds
on polypeptides, producing a color
change from blue to a deep violet
or blue color.
 Oftentimes a light pink color may
result in the presence of small
peptide chains.
 2 ml of extract mix + 1 ml of 10%
NaOH + 5 drops of copper sulfate
 Gray color: (-) protein or peptide
LAB
 LABORATORY EXPERIMENTS 1 AND 2

the enzyme molecule will begin to move

more rapidly (a.k.a. shake and shimmy).
When the enzyme “shimmies” it unfolds.
When an enzyme unfolds, it has
denatured and no longer has shape or
function. Therefore, it cannot bind to the
substrate anymore so it cannot speed
up the reaction anymore.

• Test for Specificity of Enzyme Action

o The salivary amylase is an enzyme that
catalyze the hydrolysis of starch,
yielding dextrins, then a mixture of
glucose, maltose, and maltotriose and
small branched dextrins.
o The action of a-amylase can be followed
by observing the time taken to reach the
point at which the reaction mixture no
longer gives a colour with the iodine
2. Catalase Activity solution, the 'achromic point'.
o The time required for the hydrolysis of
 5 ml of extract + 1ml of 3% H2O2 starch will be correlated to the relative
 Heated – no formation of gas enzyme activity.
 + 1ml of 0.5% benzidine o When enzyme activity is high, the time
 Result: Brown Color for the starch to hydrolyze will be very
 According to this equation short.
o When the enzyme is operating poorly or
2 H2O2 (l) → 2 H2O (l) + O2 (g) not at all, the activity is low, and more
 A gas is formed (oxygen gas) only if the time will be required for the starch to
reaction happens and the products are hydrolyze.
formed. When a gas is formed inside of
a liquid, we see the gas as bubbles.
Because we see bubbles being formed
in the test tube with potato mixture, it
indicates that the reaction took place.
The only way that the reaction could
take place at a fast-enough rate for us
to see the bubbles is, if the catalase
enzyme is there to speed up the
reaction. Therefore, catalase must be
present in potato.
 High temperature caused the catalase
to have NO ACTIVITY. (The catalase is
not functional) You know this because
you did NOT see bubbles. Therefore,
the reaction was not sped up by
catalase and products were not formed
in the lab time frame.
 At the molecular level, the catalase was
denatured by the high heat. Heat
causes the enzyme molecule to have
more kinetic energy. This means that

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D. QUESTIONS
1. What are Coenzymes?
• Coenzymes, many of which are derivatives
of B vitamins, serve as “shuttles” for
commonly used groups such as amines,
electrons, and acetyl groups. Coenzymes
are organic cofactors that help enzymes
drive chemical reactions in the body. It
temporarily binds to an enzyme to change
its shape or configuration. Once the enzyme
is in its active form, it can build or
breakdown substrates into products.
2. How do the following factors affect
enzymatic activity?
a. pH
o Increase in the hydrogen ion concentration
(pH) influences the enzyme activity.
o Extreme pH can lead to denaturation of
enzyme because the structure of the
catalytically active protein molecule
depends on the ionic character of the amino
acid side chains.
o The pH at which maximal enzyme activity is
achieved is different for different enzymes
and often reflects the [H+] at which the
enzyme functions in the body.
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LABORATORY EXPERIMENTS 1 AND 2
b. Temperature
o As temperature increase the rate of reaction
increases however beyond the point of
optimum temperature the rate of enzyme
catalyzed reaction decreases because the
hydrogen bond and the hydrophobic
interaction in the enzyme structure is being
broken.
o When this occur enzyme losses it shape
and the substrate is unable to bind properly
to the active site thus reducing enzyme
activity.
o amount of kinetic energy increases in the
system thus increasing the rate of reaction
since the collision frequency between the
substrate and the enzyme active site
increases.
o Also as temperature increases the substrate
molecules gain more energy to overcome
the activation barrier hence forming more
ES complex which alternately increases rate
of enzyme reaction.
c. Enzyme Concentration
o The rate of the reaction is directly
proportional to the enzyme concentration
provided that the condition of the reaction
remains constant and sufficient substrate is
supplied.
o For example, if the enzyme concentration is
halved, the initial rate of the reaction (Vo),
as well as that of Vmax, are reduced to half
that of the original.
3. What is the clinical relevance of measuring
transaminase (AST or ALT) in the blood?
Give the normal values for these enzymes.
o ALT and AST measurements are particularly
useful in the diagnosis and management of
certain liver diseases, e.g. viral hepatitis and
cirrhosis
o Normal values
 AST: 10-34 IU/L
 ALT: 8-20 IU/L
4. What is the clinical significance of LDH,
CPK? What are the different isotypes of CPK
and where are they found? Give the normal
values for these enzymes.
 LDH (lactate dehydrogenase) and CPK
(creatine phosphokinase) are
LAB
 LABORATORY EXPERIMENTS 1 AND 2

determined in the diagnosis of o Genetic material contains DNA while RNA is

myocardial infarction. important in the formation of new DNA and
 CPK 1 = BB – brain and smooth the translation of DNA into proteins.
 muscle CPK 2 = MB – heart A. Procedures
 CPK 3 = MM – muscle
o Normal Values 1. Isolation of RNA from Yeast
 CPK males: 38 – 174 lu/L a. Mix 2 grams of yeast with 3 grams of white
 CPK females: 76 – 140 lu/L sand. Grind the mixture in a mortar.
 MB fraction creatine kinase less b. Then add 15 ml of freshly prepared 0.2%
than 4.0 ug/mL NaOH to make a smooth creamy paste.
c. Pour the mixture in a 250 ml beaker
VI. NUCLEIC ACIDS
and the volume 50 ml by adding
• Polymers of nucleotides enough 0,2% NaOH solution.
• Constituents of nucleotides d. Cover with a watch glass to avoid
- Nitrogen-containing base (a purine or evaporation.
pyrimidine) e. Heat the beaker in a water bath controlled
- Sugar at 90C for 30 minutes.
- Phosphate f. Filter 3 times through cheese cloth and
• Mostly conjugated with proteins (e.g. histones) once through filter paper.l Cool the filtrate.
to form nucleoproteins Perform the following tests on the filtrate.
- Hydrolysis of nucleoproteins yields
proteins and nucleic acids 2. Qualitative Tests
• On further hydrolysis of the nucleic acids, a. Test for Nucleoproteins
several components are obtained as shown in  To 1ml of the filtrate in a test tube,
the diagram: 1ml of 10% NaOH solution & 5-10
drops of 1% CuSO4. Note the color
NUCLEOPROTEINS formed.
 Result: Violet
b. Mild Acid Hydrolysis
PROTEINS NUCLEIC ACIDS  Add 20 ml of 10% H2SO4 to the
remaining the remaining filtrate in a
beaker. Boil gently for a few minutes.
NUCLEOTIDES
Perform the ff. tests on portion of the
resulting solution.
 Result: Yellow precipitate
indicates presence of
PHOSPHATES NUCLEOSIDES phosphate

D-ribose Pyrimidines c. Test for the Presence of Phosphates

D-deoxyribose Purines To 1 ml of the test solution, add 2 ml
of HNO3 & 2 ml of 5% ammonium
Figure 25. Hydrolysis of Nucleoproteins. molybdate solution. Heat to boiling.
Let it stand for a few minutes.
• Nucleic Acids – may either be a DNA or Observe the color of the precipitate
RNA depending on the sugar attached to formed.
the nitrogen base.  Result: Yellow precipitate
o DNA indicates presence of phosphate
− Contains deoxyribose d. Test for the Presence of Ribose
− Has the pyrimidine known as  Prepare 3 test tubes as follows: test
thymine tube # 1-1 ml solution from the acid
o RNA hydrolysis test tube # 2-1 ml 0.1%
− Contains ribose ribose and test tube # 3-1 ml 0.1%
− Has the pyrimidine known as uracil glucose solution.

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 Add 3 ml of Bial’s Orcinol reagent to
each test tube. Place the test in a
boiling water bath until a color
develops. Compare the color formed in
test tube one with those other two
 Result: Light yellow (Clear)
e. Test for the Presence of Purines
 Add 3 ml of 10% NH4OH 2 ml of the
solution in a test tube. Mix 2-3 drops of
• 5% AgNO3 solution to it. Note the color of the
precipitate produced.
• Result: Light brown precipitate
B. Questions
1. What is the effect of the number of A-T
or G-C pairs on the melting point of
DNA?
- The melting point of DNA is influenced by
its base composition. The A-T base pairs
are held together by only 2 hydrogen
bonds, whereas in C-G base pairs, there
are 3 hydrogen bonds. Hence, a DNA that
contains high concentrations of A-T pairs
denatures at a lower temperature than a
DNA with rich G-C pairs
2. Enumerate the components of the
mononucleotide polymers of DNA and
RNA.
• DNA
o The mononucleotide polymers of DNA
include bases like AMP, GMP, TMP,
and
CMP
o DNA: adenine-adenine=AMP,
thymine-thymine=TMP, cytosine-
cytosine=CMP
• RNA
o The mononucleotide polymers of RNA
include bases like AMP, UMP, and
CMP.
o RNA: adenine, guanine, cytosine,
uracil, uridine
3. Give the method for the quantitative assay of
DNA in a given sample.
- Beer-Lambert’s equation absorbance
should be taken using a spectrometer
measured at alpha-260nm, pH 7.0. The
value of the absorbance actual
concentration and quality of DNA at given
sample will be completed
VI. PRINCIPLES OF CHROMATOGRAPHY
A. Chromatography
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LABORATORY EXPERIMENTS 1 AND 2
- A method of separating substances in a mixture.
It is a physical method of separation in which the
components to be separated are distributed
between two phases:
a. phase constituting a stationary bed of
large surface area
b. phase of being a fluid that percolates
through or along a stationary bed
- Paper Chromatography
o a type of partition chromatography
that uses paper as support for the
stationary phase, water. The
stationary and mobile phase in
paper chromatography is liquid.
Separation depends on relative
tendencies of molecules in a
mixture to associate more strongly
with one or other phase. This is
applicable not only to amino acids
but also sugars.
B. Circular Horizontal Chromatography of Amino
Acids
- It has many advantages in the lab
particularly in the simplicity of the required
apparatus, the speed of development and
the capacity of comparing different
samples at the same time.
- A string attached to the center of the disk
through a suitable cut in the center of the
paper serves as the support through which
the developing solvent will flow. Petri dish
covers make suitable chambers for
supporting the paper. The main factor,
which influence Rf values of amino acids,
with this method, are pH, type of paper,
temperature, and length of exposed wick.
- It is desirable to analyze both standard
and unknown solution on the same disk.
This is accomplished by applying
equidistant spots on a pencil-line drawn at
a radius of 1-cm from the center.
- A series of segments emerge after color
development, which is compared to similar
segment containing known compounds.
VII. NUTRITION
- Nutrition – the sum total of all biochemical
processes involved in taking in nutrients and
assimilating them via absorption and utilizing
them via metabolism in the human body.
- Classification (based on the amount taken in):
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 LABORATORY EXPERIMENTS 1 AND 2

o Macronutrients include carbohydrates, o Only when sunlight exposure is

proteins, and fats inadequate is a dietary source
o Micronutrients include vitamins and required
minerals o Functions:
•
Regulation of calcium absorption and
A. Questions homeostasis; most of its actions are
- Enumerate the four fat-soluble vitamins and mediated by way of nuclear receptors
describe their importance in human nutrition that regulate gene expression
•
It also has a role in regulating cell
• VITAMIN A
proliferation and differentiation
- Important in visual function
o Deficiency
In the retina, retinaldehyde
functions as the prosthetic group of
•
Leads to rickets in children and osteomalcia
in adults (continues to be
the light-sensitive opsin proteins,
forming rhodopsin (in rods) and a problem in northern latitudes, where
iodopsin (in cones) sunlight exposure is inadequate)
Any one cone cell o Vitamin D is Toxicity
contains only one
type of opsin and
•
This leads to contraction of blood
is sensitive to only vessels, high blood pressure, and
one calcinosis (the calcification of soft
color tissues)
o Vitamin A deficiency is the most important •
In some cases, hypercalcemia in response to
preventable cause of blindness low intakes of vitamin D is due to genetic
•
Earliest sign of deficiency is a loss of defects of calcidiol 24-hydroxylase, the
enzyme that leads to inactivation of the
sensitivity to green light, followed by
vitamin
impairment to adapt to dim light, then
night blindness
•
Although excess dietary vitamin D is toxic,
excessive exposure to sunlight does not
•
More prolonged deficiency leads to
lead to vitamin D poisoning, because there
xerophthalmia: keratinization of the
is a limited capacity to form the precursor, 7-
cornea, and blindness
dehydrrocholesterol, and prolonged
o Vitamin A has an important role in
exposure of previtamin D to sunlight leads
differentiation of immune system cells, and to formation of inactive compounds
even mild deficiency leads to increased
susceptibility to infectious diseases
VITAMIN E
o Vitamin A is toxicity
o Functions:
•
This leads to •
Vitamin E does not have a
accumulation beyond precisely defined metabolic
the capacity of function
intracellular binding It acts as a lipid-soluble
proteins; unbound
vitamin A causes antioxidant in cell membrane,
membrane lysis and where many of its functions can be
tissue damage. provided by synthetic antioxidants,
•
Symptoms include: and is important in maintaining the
−
Affects the central nervous system fluidity of cell
(headache, nausea, ataxia, and membranes
anorexia, all associated with increased o Main function of vitamin E - is as a
CSF pressure);
chain-breaking, free-radical-trapping
−
Liver (hepatomegaly with histological
changes and hyperlipidemia); antioxidant in cell membranes and
−
Calcium homeostasis (thickening of the plasma lipoproteins by reacting with
long bones, hypercalcemia, and the lipid peroxide radicals formed by
calcification of soft tissues); and the peroxidation of polyunsaturated fatty
−
Skin (excessive dryness, desquamation, acids
and alopecia) o It also has a (relatively poorly defined)
role in cell signaling
VITAMIN D o Deficiency
o Not only that vitamin D is synthesized in o Results in resorption of
fetuses and testicular
the skin, but it is also considered as a atrophy
hormone o Dietary deficiency of vitamin
E in human beings is

TRANSCRIBERS
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22 | 23 EDITORS
 LABORATORY EXPERIMENTS 1 AND 2

unknown, although patients o Next, calculate percentage of

with severe fat total kcal:
malabsorption, cystic o 100 grams of protein x 4 kcal/g = 400 kcal
fibrosis, and some forms of o 700 grams of CHO x 4 kcal/g = 2800 kcal
chronic liver disease suffer o 80 grams of fat x 9 kcal/g = 720 kcal
deficiency because they o 20 grams of alcohol x 7 kcal/g = 140 kcal
were unable to absorb the o Calculate the total kcal:
vitamin or transport it, o 400 + 2800 + 720 + 140 = 4,060 kcal
exhibiting nerve and muscle
membrane damage REFERENCES:
o Premature infants are
born with inadequate 1. Previous Trans
reserves of the vitamin
o The erythrocyte
membranes are
abnormally fragile as a
result of lipid
peroxidation, leading to
hemolytic anemia
• VITAMIN K
o Functions
o required for
synthesis of blood-
clotting proteins
o Three compounds have the biological activity
of vitamin K:
•
Phylloquinone, the normal
dietary source, found in green
vegetables;
•
Menaquinones, synthesized by
intestinal bacteria, with differing
lengths of side chain; and
•
Menadione and menadiol diacetate,
synthetic compounds that can be
metabolized to phylloquinone
o Vitamin K is the coenzyme for carboxylation of
glutamate in post-synthetic modification of
calcium-binding proteins
o Vitamin K is also
important in synthesis
of bond and other
calcium-binding
proteins

2. Compute for the amount of Kcal

in the following diet:
• 100 grams of protein
• 700 grams of CHO
• 80 grams of fat
• 20 grams of alcohol

o First, identify the value of

energy-yield nutrients, these
are:
o Carbohydrate – 4 kcal/g o
Protein – 4 kcal/g
o Fat – 9 kcal/g
o Alcohol – 7 kcal/g

TRANSCRIBERS
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23 | 23 EDITORS