Differential Centrifugation

1st Centrifugation – Sediment 1: Unbroken cells, nuclei

Major biomolecule: DNA – Nucleic Acid
2nd Centrifugation – Sediment 2: Mitochondria, lysosomes, peroxisomes
Major biomolecule: Carbohydrates
3rd Centrifugation – Sediment 3: Microsome, and other small vesicles
Major biomolecule: Lipids, proteins
Supernatant 3: Proteins and some inorganic ions
Chemical Reaction
■ Diphenylamine solution – a mixture of diphenylamine, glacial acetic acid
and sulfuric acid

Chemical Reaction
■ The reaction is a formation of complex between copper and oxygen and
between copper and nitrogen
Chemical Reaction

● The reaction is a dehydration reaction forming a furfural compound. The α-

naphthol forms a complex that gives the positive sample a purple ring
 Enzymes Experiment 3A MC-2 LAB
OBJECTIVES of the Experiment
❏ Demonstrate the catalytic action of enzymes through different organic
specimen
❏ Recognize the specificity of enzymes to its substrate
❏ Identify the products of each reaction through different color tests
❏ Describe the role of enzyme in digestive processes

ENZYMES
❏ Enzymes are substances that act as a catalyst for biochemical reactions by
finding a pathway which has lower activation energy.
❏ Each enzyme has an active site where the chemical reactions occur. The
reactant of the said chemical reaction is called substrate.
❏ CHARACTERISTICS

 They are not altered or consumed during reaction.

 Reusable
 Enzymes show specificity to the reaction they control
 Enzymes are sensitive to their environment so they can be controlled
by adjusting the temperature, the pH or the substrate concentration

Pepsin
❑ an enzyme that hydrolyzes protein to smaller peptides to amino acids,
cleaving peptide bonds between hydrophobic and aromatic amino acids.
❑ it is active in acidic environment, having an optimum pH of 1.5-2.0
❑ Proenzyme pepsinogen, is released by the chief cells in the stomach wall.
For it to be activated it reacts with hydrochloric acid of the gastric juice and
become pepsin.
Amylase
❑ Salivary Amylase (Ptyalin)
❑ Present in saliva with optimum pH of 6.7-7.0
❑ an enzyme that hydrolyzes α-1,4-glycosidic bonds in starch producing
oligosaccharides, maltose or glucose depending on the time spent in the
mouth of the food.

❑ Iodine Test
➢ Iodine reacts with starch forming a blue- black solution
Catalase
❑ an enzyme that protects organisms from the effects of hydrogen peroxide
❑ H2O2 is a powerful oxidizing agent and is potentially damaging to cells
❑ It breaks down hydrogen peroxide into water and oxygen gas
❑ 2H2O2 → 2H2O + O2
❑ Potato catalase - enzyme found in potato, optimum pH = 9.0

Factors Affecting Enzyme Activity Experiment 3B MC-2 LAB

OBJECTIVES of the Experiment
❏ Describe the effect of the factors presented in the experiment on
enzyme’s activity.
❏ Relate the factors to human enzymatic metabolic activities .

Enzymatic Action
❏ Catalysis of enzymes can increase the reaction rate many million times
faster than without a catalyst
❏ Their action on the substrate can be controlled by adjusting the
temperature, pH, or substrate or enzyme’s concentration.
❏ Further, since they are proteins, they also affected by agents that causes
them to undergo denaturation. This experiment shows the effect of these
factors on enzymatic reactions.
Effect of temperature

❏ Temperature increases enzyme activity up to its optimum temperature

❏ Optimum temperature is the temperature at which enzymatic reaction
occur fastest
❏ For most enzymes, the optimum temperature is about 40°C

Effect of Ph
❑ The effect of pH depends on the enzyme’s optimum pH (pH where enzyme
is most active). But extreme pH levels will cause denaturation.
❑ If pH slightly different from the enzyme’s optimum value, there will be
small changes in the charges of the enzyme and its substrate’s molecule.
❑ This change in ionization will affect the binding of the substrate with the
active site.
❑ Salivary amylase has an optimum pH of 6.8

Effect of Enzyme Concentration

❑ When enzyme concentration is continuously increasing, as shown in the

top image, the reaction rate will continue to increase.

❑ Increasing enzyme concentration will increase rate of reaction but when
enzyme is no longer available, it reaches a maximum point (bottom image).
Effect of Substrate Concentration
❑ The rate of reaction increases as substrate concentration increases (at
constant enzyme concentration)
❑ The increase in velocity is proportional to the substrate concentration but
when enzyme is no longer available, V max is reached – saturation point

Enzymes Experiment 4A MC-2 LAB

OBJECTIVES of the Experiment
❏ Extract DNA from plant tissue
❏ Describe each procedure done during the extraction
❏ Analyze the extracted DNA through a qualitative test

NUCLEIC ACID
❏ A nucleic acid is an unbranched polymer in which the monomer units are
nucleotides.
❏ Types
❏ DNA stores the genetic information of an organism and transmits that
information from one generation to another.
❏ RNA translates the genetic information contained in DNA into proteins
needed for all cellular function.
DNA
❑ DNA is located in the nucleus of the cell
❑ RNA is located in different parts of the cell

 hnRNA in nucleus
 mRNA in cytoplasm
 rRNA in ribosome
 tRNA in cytoplasm

DNA Extraction
STEPS
1. Breaking of cell wall (if sample is plant) – done by mechanical disruption
through crushing or blending
2. Breaking of cell membrane to expose organelles – done by adding the
dishwashing liquid which is an amphipathic substance
▪ The long hydrocarbon tail of soap breaks the lipid bilayer of cell membrane
while the soap’s polar head is attracted to the water
3. Salt binds to the negatively charges phosphate end of the DNA chain to
protect the chain as well as increases the separation of DNA from the
hydrophobic layer
4. Filtration removes cellular debris from the sample
5. Precipitation with cold alcohol
▪ DNA becomes insoluble in water in the presence of salt and alcohol (more
polar)
▪ DNA precipitate as a white gelatinous material
 HYDROLYSIS OF NUCLEIC ACID Experiment 4B MC-2 LAB
OBJECTIVES of the Experiment
❏ To hydrolyze DNA sample
❏ To qualitative test the products of hydrolyzed DNA sample
❏ Differentiate unhydrolyzed DNA to acid hydrolyzed DNA

REVIEW
● Nucleotide is composed of base, sugar and phosphate group
● Glycosidic bond joins the base and the sugar while phosphodiester bond
joins the phosphate and sugar group
● The nucleotide chain is joined together by phosphodiester bond.
● Not affected by addition of base

HYDROLYSIS
Acid Hydrolysis (Lysis – break down)
❏ Depurination
❏ break down of glycosidic bonds only if pH > 3
❏ Products are purine base and deoxyribose with phosphate group

❏ Total breakdown
❏ break down of glycosidic bond with phosphodiester bond if pH < 2 plus
heat
❏ Extremely low pH hydrolyses DNA completely
❏ Products are phosphate group, the purine or pyrimidine base, and
deoxyribose

Acid Hydrolysis
❏ Reactions starts with an acid attacking the hydrogen bonding between
complementary bases
❏ Further hydrolysis breaks the nucleobases specifically the purines
❏ This happens because it is easy to attack the glycosidic bond than the
phosphodiester bond
Test for Purine Bases
❑ Test for the presence of Adenine and Guanine
❑ Reagents: Silver Nitrate and Ammonium Hydroxide
❑ Silver ion in ammoniacal solution precipitates purine bases reacting to the
nitrogen of purine base
❑ Positive result: gelatinous white precipitate
 LIPIDS Experiment 5A MC-2 LAB
OBJECTIVES of the Experiment

 Observe the different properties of different lipid samples

 Learn how to characterize lipids through different tests
 Identify the type of lipids based on chemical and physical properties

LIPIDS

 One of the major biomolecules in living cells that have no common

structure unlike proteins, carbohydrates, and nucleic acids.
 Lipids are defined as organic substances that are insoluble (or
sparingly soluble) in water but soluble in organic solvents.
 This physical characteristic (solubility) as well as the chemical
properties of lipids depends on the presence of carboxyl groups,
number of double bonds, number of hydroxyl groups, and length of
carbon chains.

TYPES

 SIMPLE - are esters of fatty acids and alcohol

 Triglycerides (Fats and Oils)
- esters of fatty acids and glycerol
 Waxes - esters of fatty acids and monohydric alcohol
 COMPOUND - are esters of fatty acids and alcohol that contain
another functional group
 Phospholipids - esters of fatty acids with glycerol containing
an esterified phosphoric acid and a nitrogen
 Glycolipids - contain an amino alcohol (sphingosine or iso-
sphingosine) attached with an amide linkage to a fatty acid
and glycosidically to a carbohydrate (sugars, amino sugar,
sialic acid)
 ANALYSIS OF EGG LIPIDS Experiment 5B MC-2 LAB
OBJECTIVES of the Experiment

 Extract lipids from egg yolk

 Identify the extracted lipids through different tests

Extraction of Lipids

 Characterization of lipids depend on its solubility in organic solvents,

immiscibility with water, physical characteristics (relatively low
density)
 Principle of lipid extraction depends on the polarity of the lipids
present compared to the polarity of the solvent.
 Polar lipids (such as glycolipids or phospholipids) are more soluble in
polar solvents (such as ethanol or acetone),
 Non-polar lipids (such as triacylglycerols) are more soluble in non-
polar solvents (such as hexane and cylohexane) than in polar ones.
 Lipids vary in polarities hence it is very unlikely to select a single
organic solvent to extract them all

Yolk Composition

 Egg yolk is a mixture of lipoproteins.

 Protein content of yolk is about 16% and lipid content varies from
32% to 35%,
 Yolk lipid fraction is made up of:
  66% triglycerides, 28% phospholipid, 5% cholesterol, small amounts
of other lipids.
 Cholesterol in the egg is found only in the yolk.
 Egg yolks contain Lecithin, a a glycerophospholipid, containing fatty
acids, glycerol, phosphate, and choline

Yolk Composition

 Lipids in egg are concentrated in the yolk. One-third of the yolk is

lipid, which consists of about two-thirds neutral lipids and one-third
polar lipids.
 The neutral lipids are composed of sterolester, carotenoid,
triglyceride, fatty acid, diglyceride, sterol triglyceride is predominant
 Position 1 of the triglyceride molecules is mostly occupied by palmitic
acid (saturated acid), position 2 by oleic and linoleic acids
(unsaturated acids);

Simple Extraction of Lipids

STEP 1 – Addition of salt – this crudly removes proteins from the lipid portion
of the egg yolk
STEP 2 – Addition of cyclohexane and ethanol – this process separates the
polar and nonpolar lipids
STEP 3 – Addition of acetone – this process removes the slightly polar to
highly polar lipids
QUALITATIVE TESTS FOR CARBOHYDRATES Experiment 6
MC-2 LAB
OBJECTIVES of the Experiment

 Perform the different tests for different types of carbohydrates

 Understand the principle behind each test and its purpose
 Identify the unknown carbohydrate based on the results of the tests

CARBOHYDRATES

 Carbohydrates are biomolecules that is compose of carbon, hydrogen

and oxygen atoms whose functional group can either be aldehyde or
ketone.
 It can be classified into monosaccharide, the monomer unit of
carbohydrates, disaccharide if two monosaccharides are joined,
oligosaccharide if few monosaccharides are joined or polysaccharide
if it is already composed of large number of monosaccharides.
MOORE’s Test
 Test for reducing sugars
 Principle: Reducing sugar undergoes aldol condensation
(caramelization reaction) under alkaline/basic conditions because of
the free anomeric carbon
 positive for: ribose, galactose, glucose, maltose, fructose,
lactose
 negative for: glycogen, sucrose and starch
 Positive Result: Caramel odor and brown-yellowish colored complex
other positive results: yellow/orange/dark brown + caramel odor