Experiment number 1: PROTEINS Quaternary Structure – exists in proteins

containing more than one polypeptide chains

Terms: such as hemoglobin.
Protein – complex biomolecules composed of Protein Denaturation - a process in which
alpha amino acids joined by peptide linkages. proteins or nucleic acids' secondary, tertiary
Peptide bond – is a carbon to nitrogen bond and quaternary structures are disrupted.
(an amide bond) formed by the loss of a
molecule of H2O from the –COOH group of one
amino acid and the –NH2 group of an adjacent
amino acid.

Amino acid – carboxylic acids with an amino

group attached to the alpha carbon. Physical and Chemical Agents That Are Capable
of Denaturing Proteins:
 20 amino acids found in nature are a-amino
acids except proline. Physical Agents:
 Each amino acid has a unique property due
to the composition of its R group. Heat

Zwitterion – amino acids in the solid state exist  Can be used to disrupt hydrogen bonds
and non-polar hydrophobic proteins.
Isoelectric point – The PH at which the number  This occurs because heat increases the
of positive and negative charges in an amino kinetic energy and causes the
acid or a protein are the same. molecules to vibrate so rapidly and
violently that the bonds are disrupted.
Proteins have complex structures that are
 The proteins in eggs denature and
described at four levels:
coagulate during cooking.
Primary Structure – linear sequence of amino
Microwave radiation
acids held together by peptide bonds.
 Causes violent vibrations of molecules
Secondary Structure – result of regular folding
patterns of specific regions of the polypeptide that disrupt hydrogen bonds
chain. It is maintained by hydrogen bonding Ultraviolet radiation
between the amide hydrogen of one peptide
bond and the carbonyl oxygen of another  Operates very similarly to the action of
peptide bond. heat (e.g. sunburning)

Tertiary Structure – the three-dimensional Violent whipping or shaking

structure that results from attractive forces
among the side (R) chains.  Causes molecules in globular shapes to
extend to longer lengths, which then
entangle
Chemical Agents: How Protein Denaturation Can Be Used in
Medical Field
Detergent
 It may be used for sterilization,
 Affects R-group interactions disinfectant, and digestion. Medical
Organic Solvents (e.g., ethanol, 2-propanol, instruments could be sterilized, toxins
acetone) could be disinfected and the protein
digestion could occur for muscle
 Interferes with R-group interactions formation.
because these solvents also can form
hydrogen bonds; quickly denatures
proteins in bacteria, killing them.

 The 70% ethyl alcohol is the most

effective as a disinfectant because the
concentration is the most able to
invade the bacteria cell wall and
denature the proteins and enzymes
inside the cell. Less dry than isopropyl. A. Protein Denaturation

Strong acids and bases Objective of the Experiment

 Disrupts hydrogen bonds and salt a. To observe the effects of several

bridges; prolonged action leads to denaturing agents on a protein sample.
actual hydrolysis of peptide bonds b. To observe the effect of heavy metal
ions (reagent) on a protein sample.
Salts of Heavy Metals (e.g., salts of Hg2+, Ag+,
Pb2+) Procedures

 Metal ions combine with –SH groups EFFECT OF HEAT AND ALCOHOL
and form poisonous salts
a. Place 2.0 mL of egg white solution in a
Amino Acid in a Protein That Are Reactive With test tube and heat in a boiling water
Heavy Metals bath for 5 minutes.
b. Label two tubes as 1 and 2. Add 2.0 mL
 Heavy metals are high molecule of egg white solution to each tube. To
weighted, salts are ionic and disrupts tube 1, add 95% ethanol and to tube 2,
salt bridges in proteins. Thus, leads to 70% ethanol. Compare the appearance
an insoluble metal protein salt. For of the resulting mixtures with the
example, cysteine. standard.

Reducing Agents EFFECT OF HEAVY METALS

 Reduces disulfide linkages to produce a. Add 2.0 mL of egg white solution to

-SH groups each of the two test tubes labelled 1.
 b. To test tube 1, add 1.0 mL of 1% AgNo3 Objective of the Experiment
solution. Mix well and note the color of
the precipitates formed. Set aside for 5 a. To show the positivity for aromatic
mins. groups. (tyr, trp, phe)
c. Decant the supernatant liquid and test Procedures
the solubility of a small portion of the
precipitate in 5.0 mL of water. a. Add 0.5 mL of concentrated HNO3 to
1.o mL of egg white solution in a test
Experimental Results tube.
Effect of Hat and Alcohol b. Mix with a stirring rod and warm in
water bath for 5 minutes. Note the
Observations color of precipitate formed.
c. Cool the contents of the tube and make
it alkaline by adding 50% NaOH. Note
Heat Heavy Precipitation
the changes in the mixture.
(++)
Experimental Results
95% Ethanol No Precipitation (-)

Color obtained
70% Ethanol Slight Precipitation
(+)
With HNO3 Yelow precipitate

With NaOH Orange

Effect of Heavy Metals

Color Solubility
C. Biuret Test

AgNO3 Dirty White Insoluble  Peptide Bond - a chemical bond formed

between two molecules when the
carboxyl group of one molecule reacts
with the amino group of the other
B. Xanthoproteic Test
molecule, releasing a molecule of water
(H2O).
 Yellowish substance produced by
heating protein with nitric acid is
xanthoprotein, hence the name
xanthoproteic for the test. Objective of the Experiment
 This test is performed to identify the
a. To find out if there’s presence of
amino acid having benzene ring in them
proteins and peptide bonds in the
like tyrosine, tryptophan, and
solution.
phenylalanine.
Procedures Experimental Results

2.1
Positive result Negative result
a. Mix 1.0 mL of egg white solution and 10
gtts of 6M NaOH in a test tube. Violet Blue
b. Add 1 drop of 0.5% cuSO4 solution.
c. Dissolve one tablet of aspartame or ½
Reagents:
sachet of Equal in 2.0 mL of water
 Egg white solution, 6M NaOH, 0.5%
d. Add to the resulting solution 10 gtts of CuSO4, and Aspartame
6M NaOH and 0.5% CuSO4 slution.  Blood plasma, 23% Na2SO4, and Ether

2.2

a. Label one test tube TP (Total Protein),

D. Ferric Chloride Test
then add 3 gtts of blood plasma (or
 Phenylalanine specific amino acid that
serum) and 3.0 mL of 23% Na2SO4
can be seen in the test
solution. Mix well.
b. Transfer half of the mixture from the  Phenols - similar to alcohols but form
tube marked TP to the centrifuge tube. stronger hydrogen bonds. Thus, they
Set aside the tube marked TP. are more soluble in water than are
c. Add 1.0 mL of ether to the mixture in alcohols and have higher boiling points.
the centrifuge tube, mix thoroughly, Phenols occur either as
and stopper it. Centrifuge for minutes. colourless liquids or white solids at
Note the color of the precipitate room temperature and may be highly
formed at the junction of the two toxic and caustic.
liquids. Objective of the Experiment
d. Using very thin pipette, carefully draw
out the lower aqueous layer, taking a. Its purpose is to detect the presence or
care not to disturb the precipitate at absence of phenols.
the interface. Wipe the pipette to
Experimental Results
remove any precipitate that adheres to
it.
e. Transfer the contents of the pipette to a Color obtained
test tube and mark it as A (albumin)
f. To each of the two tubes (TP and A),
Golden Yellow
add 1.0 mL of 6M NaOH and 2gtts of
0.5% CuSO4 solution.
Reagents: Milk, FeCl3
E. Hopkins-Cole Test Experimental Results
 Tryptophan - It is an essential amino
acid. Color obtained

Aspartame purple

Egg white Solution pink

Objective of the Experiment

Reagents: Ninhydrin Solution, egg white
It detects the presence of tryptophan in solution and Aspartame

proteins

Experimental Results
G. Sakaguchi Test
Color obtained  a-naphthol solution and sodium
hypochlorite (bleach) – is a sakaguchi
reagents (oxidizing agents)
Violet Ring
 NaOH (sodium hydroxide) – hydrolyzing
agent
Reagents: Hopkins-Cole Reagent, egg
 The sakaguchi reagents reacts with
white solution, and H2SO4
NaOH that will give off red
coloration/precipitate
F. Ninhydrin Test  Egg white is made up of protein. To
 Amino Acid - contains Amino (NH2) detect specific amino acids in the we
Carboxyl (COOH) groups. need to break down complex structure
 Proline and hydroxyproline are the of its protein.
only amino acid that doesn’t contain  NaOH is a hydrolyzing agent that helps
free amino acid you breakdown proteins into amino
acids.
Objective of the Experiment  Sakaguchi reagents will react on the
guanidinium group of arginine causing a
 used also to detect free amino and
red precipitate.
carboxylic acid groups on proteins and
 Shoyo Sakaguchi (1925) – a first
peptides.
technologies and a chemist. First
conducted the sakaguchi test.

Guanidine

 is the compound with the formula

HNC(NH2)2. It is a colourless solid that
dissolves in polar solvents. It is a strong
base that is used in the production
 of plastics and explosives. It is found d. Immediately note the color of the
in urine as a normal product of resulting solution as it fades quickly.
protein metabolism. A Record your observation.
guanidine moietyalso appears in larger Experimental Results
organic molecules, including on the side
chain of arginine.
Positive result Negative result
Arginine
Red precipitate No precipitate
 The amino acid side-chain of arginine
consists of a 3-carbon aliphatic straight
chain, the distal end of which is capped Reagents: Egg white solution, 10% NaOH,
by a guanidiniumgroup, which has 0.2% a-napthol solution and sodium
a pKa of 12.48, and is therefore always hypochlorite

protonated and positively charged at

physiological pH. Because of
a. What is the principle involved in this
the conjugation between the double
test? What is it’s purpose?
bond and the nitrogen lone pairs, the
- This test is for the detection of a
positive charge is delocalized, enabling specific type of protein with the
the formation of multiple hydrogen amino acid containing the
bonds. Polar basic amino acid that guanidinium group, this test is used
consist of two amino group and one to detect the presence of arginine.
carboxyl group and the second amino b. Why should NaOH be added first before
acid is part of its side chain. the other reagents? Illustrate with an
equation the effect of adding NaOH to
Objective of the Experiment arginine.
- Hydrolysis of egg white proteins
 Detection of a specific type of protein
breaks down the larger
with the amino acid containing the
molecules into smaller
guanidinium group, this test is used to molecules for easier digestion.
detect the presence of arginine. c. What is the purpose of the other
reagents?
Procedures
- Sakaguchi reagent consists of 1-
a. Strictly observe the order of addition of Naphthol and a drop of sodium
the reagents. To the 5.0 mL of egg hydroxide it reacts with the arginine
white solution, add 1.0 mL of 10% in the egg white solution.
NaOH solution. d. What group in arginine responds to this
b. Mix, then add 1,0 mL of 0.2% α – test?
napthol solution. Mix thoroughly. - The guanidine group in arginine
c. After 3 minutes, add 5 drops of sodium reacts with Sakaguchi reagent to
hypochlorite (bleach). form a red-coloured complex.
Oxidizing agent: An oxidizing agent, or oxidant, HCL
gains electrons and is reduced in a chemical
reaction. Also known as the electron acceptor, Hydrochloric acid, also called HCl, is a clear,
the oxidizing agent is normally in one of its highly corrosive liquid. HCl is one of the many
chemicals released in our stomach when we eat
higher possible oxidation states because it will
gain electrons and be reduced. Reduction is the a meal. The role of hydrochloric acid in the
process of gaining one or more electrons. In stomach, along with the other gastric juices, is
an oxidation-reduction, or redox, reaction, one to break down foods and cause the release of
atom or compound will steal electrons from enzymes that further aid digestion. HCl also
another atom or compound. A classic example protects the body from illness by killing
of a redox reaction is rusting. When pathogens commonly found on foods.
rusting happens, oxygen steals electrons from Pepsin
iron.
In the digestive tract pepsin effects only partial
-Hydrolysis : the chemical breakdown of a degradation of proteins into smaller units called
compound due to reaction with water. this is peptides, which then either are absorbed from
achieved by breaking a covalent bond in the the intestine into the bloodstream or are
compound by inserting a water molecule across broken down further by pancreatic enzymes.
the bond. The opposite of this is a dehydration-
condensationreaction. Small amounts of pepsin pass from the stomach
into the bloodstream, where it breaks down
Purposes of each solutions: some of the larger, or still partially undigested,
fragments of protein that may have been
CuSO4
absorbed by the small intestine.
The complex of Cu2+ ions and nitrogen atoms
make the color of CuSO4 solution changes from
blue to violet.

This color change is dependent on the number

of peptide bonds in the solution, so the more
protein, the more intense the change.

NaOH

The NaOH is there to raise the pH of the

solution to alkaline levels; the crucial
component is the copper II ion (Cu2+) from the
CuSO4.

When peptide bonds are present in this alkaline

solution, the Cu2+ions will form a coordination
complex with 4 nitrogen atoms from peptide
bonds.
Experiment number 2: ENZYMES Salivary Digestion: Factors Affecting Enzymatic
Activity
Terms:

Enzyme – special proteins that catalyze A. Effect of Temperature

biological reactions by lowering the activation
energy of a reaction. Objective of the Experiment

Substrate – reactant broken down by an  To know the effects of temperature on

enzyme enzyme activity.

Active Site – portion of the molecule Procedures

responsible for its catalytic action.
Experimental Results
Properties of and enzyme:
Temperature Color of Rank
 Absolute specific – acts on only one
solution
substrate
 Group specific – pepsin will hydrolyze
Ice Water Bath Dark blue (No 3
only soluble native proteins
Digestion)
 Linkage specific – breaks the bond only
between specific groups 37 Degrees Celsius Red (Partial 2
 Reaction specific – hydrolases, oxido- Digestion)
reductases, polymerases
Boiling Water Bath Colorless, 1
White
Apoenzyme – either simple protein or protein (Complete
Cofactor – non-protein unit Digestion)

Coenzyme – If the cofactor is an organic unit. Reagent: 1% Starch Solution, Saliva and Iodine
Metal-ion activator – If the cofactor is a metal- Solution

ion
Haloenzyme – protein and a non-protein B. Effect of pH
Digestion – hydrolysis of proteins to amino
acids, starches to monosaccharaides, and of fats Objective of the Experiment
to fatty acids and glycerol.
 To observe and identify the effects of
pH on the activity of enzyme amylase
 Mouth – Esophagus – Stomach – Small
along with the degree of digestion.
Intestine (duodenum, jejunum, and
ileum) – Large Intestine
Experimental Results Experimental Results

Temperature Color of solution Rank Test Tube Initial Color Of Time And Rank
Solution

pH 4 Red 2 1 Dark blue Control group

pH 7 Light Green 1 2 Blue 2 minutes (3)

pH 10 Dark Blue 3 3 Violet 1 minute and 30

seconds (2)

Reagents: Starch Solution, Buffer solution,

Saliva and Iodine solution 4 Light violet 25 seconds (1)

Reagent: 1% Starch Solution, Saliva and Iodine

Solution

C. Effect of Enzyme Concentration

D. Effect of Substrate Concentration

Objective of the Experiment

Objective of the Experiment
 To determine and investigate how
 To know the effect of substrate
enzyme concentration can affect the
concentration on the enzymatic activity.
initial rate of reaction.
Experimental Results  To note the color of the resulting
solution and precipitate.
Test Tube Color Of Solution RANK
Or Precipitate
Experimental Results
1 Blue 3

Test Tube Color Of Solution RANK

2 Yellow 2 Or Precipitate

3 Green 2
Saliva Negative (blue Negative (blue)
solution)

4 Red 1 Pancreatin Positive (green) Negative (blue)

and Sodiium
Reagent: 1% Starch solution, Distilled water and Carbonate
Saliva
Reagents: 1% Starch solution, Saliva, 5%
Pancreatin, 0.5% Sodium bicarbonate solution,
E. Effect of Metal-ion Poisons on Enzyme Benedict’s reagent and Barfoed’s reagent
Activity

Objective of the Experiment B. Pancreatic Proteases

 To know the effects of metal-ion Objective of the Experiment

poisons on enzyme activity.
 To compare the intensity of the colored
 Experimental Results
solutions produced
Test Tube Color Of Solution Rank
Experimental Results
Or Precipitate

Test Tube 1 Dark blue No digestion

(Vial 4)
Test Tube Color And Intensity Of
Test Tube 3 Colorless, White Complete Solution
(Vial 6) digestion

No Dark Yellow
enzyme
Pancreatic and Biliary Digestion present

A. Pancreatic Amylase Pepsin Light Yellow

Objective of the Experiment Pancreatin Light Yellow/Clear Yellow

Reagents: Egg white solution, CuSO4, 3N

NaOH, 3N HCl, Pepsin and Pancreatin
C. Pancreatic Lipase and base

Objective of the Experiment

 To know the pH level of digestion in

enzyme activity.

Experimental Results

Test Tube 1 2 3

Initial pH 1 5 6

Final pH 4 7 4

Reagents: Vegetable oil, Pancreatin, Sodium choleate,

Distilled water and 0.1% NaOH