The Human Body:

Organic Components

BIOMOLECULES
 proteins
 carbohydrates
 lipids
 nucleic acids
NITROGENOUS NON-PROTEIN
 coenzymes (vitamins)
 amine hormones
Distribution of biomolecules in a cell

http://www.aw-bc.com/mathews/
From Neil Campbell, Jane Reece, and Larry Mitchell, Biology, 5th ed.
(Menlo Park, CA: Addison Wesley Longman, 1999) © Addison Wesley Longman, Inc.
Steps for Subcellular Fractionation
1. Extraction
- first step toward isolating a specific
organelle
- most organelles and many biomolecules are
labile and subject to loss of biologic activities
- they must be extracted using mild conditions
- Cell disruption procedures are carried out at
0-4 ˚C (or in cold temperature)
- significant losses of activity can occur at
room temp. due to the action of digestive
enzyme liberated when cells are disrupted
Steps for Subcellular Fractionation
2. Homogenization
- to extract an organelle (or biomolecules)
from cells, it is necessary to disrupt the cells
under mild condition
- Most common method is by using a blender
or homogenizer
- Gentlest procedure is by hand grinding
using a mortar and pestle

Homogenate- the resulting suspension, containing

many intact organelles
Steps for Subcellular Fractionation
3. Centrifugation
- separates the homogenates into fractions
- cell organelles of different sizes are
separated according to their rates of
sedimentation in a centrifugal field
Heavier organelles sediment or precipitate
at lower speeds (and length of time)
mince
 Molisch’s Test by: Hans Molisch
(α-naphthol)

 carbohydrates are dehydrated

by H2SO4 to form an aldehyde
 aldehydes condenses with
molecules of α-naphthol
forming a furfural (violet or red
compound) which is seen in the
junction of the two liquids
 Biuret Test
(Cu++ ion in citrate solution)

 Test used to detect peptide

bonds
 Cu++ ions is reduced to Cu+
 Copper ions forms a complex
with the Nitrogen and Carbon
of the peptide bond in an
alkaline solution
 Pink to violet coloration is an
indication of a positive test
 Sudan IV

 is a chemical that is fat soluble.

 When lipids are present in

solution, it dissolves and stains
the lipid and forms a separate
reddish-orange layer.
 Dische (diphenylamine) test

• Dische reaction distinguishes

DNA from RNA with its
reaction to the pentose portion
of the nucleotide
• The sugar portion of RNA is a
ribose, while that of DNA is a
deoxyribose.
• Dische reacts with the
deoxyribose to form a blue –
colored complex
 Feulgen’s Test

 Purine base of the DNA is

removed using
hydrochloric acid.

 When the aldehyde

portion is exposed schiff’s
reagent is added resulting
to a magenta or reddish-
purple colored compound
 Orcinol test

 Is a tests for pentoses in RNA.

 Test reagent dehydrates the
pentoses to form furfural which
reacts with orcinol.
 The iron ion in the test reagent
will produce a green coloration
to bluish and precipitate may
also form.
Qualitative tests for the biomolecules
BIOMOLECULE TEST + RESULT
Carbohydrates Molisch test Violet ring between 2 liquids

Proteins Biuret test Violet or pink solution

Lipids (using Sudan IV reagent) Reddish-orange coloration

DNA Dische reaction Blue solution

DNA Feulgen’s reaction Reddish-purple coloration

RNA Orcinol test Green coloration
 Sediment 1 Sediment 2 Sediment 3 Supernate 3

Test for OBSERVATIONS/RESULTS

Carbohydrates Violet ring Violet ring Violet ring Violet ring

(Molisch test) (+++) (++) (+) (++++)
Proteins Violet solution Violet solution Light violet Dark violet
(Biuret test) (+++) (++) solution (++) solution (++++)
Lipids Red coloration Red coloration Red coloration Red coloration
(++++) (++) (+) (+)
DNA Dark blue Dark blue Blue solution Blue solution
(Dische solution (++++) solution (+++) (+) (++)
reaction)
DNA Reddish-purple Red solution Red solution Red solution
(Feulgen’s solution (++++) (+++) (+) (+)
reaction)
RNA Dark green Dark green Green solution Green solution
(Orcinol test) solution (++++) solution (+++) (+) (+)
 Experiment
PROTEINS
Objectives
1. To be able to make visual observations on the
effects of some physical and chemical agents
on proteins.

2. To be able to perform some color tests on

proteins and amino acids.
 Proteins
Proteins – high molecular mass polymers
containing the elements C, H, O and N.
Proteins – chief constituent of of all cells
of the body.
Proteins – serves to build new cells, maintain
existing cells and replace old cells
in the body.
Proteins – is necessary for the formation of
the enzymes and hormones in the body.
Proteins – yields energy in the amount of 4 kcal/g
during oxidation.
 Albumin
• Group of proteins characterized by heat
coagulability and solubility in dilute salt
solution
• Ovalalbumin (egg albumin), serum
albumin, lactoalbumin, grains and soybean
albumin
• Found in nearly all living body tissues
• Ovalalbumin
– 75% of egg white
 Denaturation
Refers to the unfolding and rearrangement
of the secondary and tertiary structures of
protein without breaking peptide bonds

For mild condition, protein can be restored

to its original conformation by carefully
reversing the conditions that caused the
denaturation called reversible denaturation.
 Consequences of Denaturation
• Decrease or Loss of biological properties
•There may also change in
– Particle size
– Increased viscosity of globular protein
– Increase in surface tension,
– Increased reactivity of side chains group
– Increased susceptibility to hydrolysis
– Change in optical rotation towards
levorotation
 Agents of Denaturation
1. Physical means
1.1 Heat 1.3 very high pressure
1.2 Violent shaking 1.4 ultraviolet radiation

2. Chemical means
2.1 Extremes in pH
2.2 High salt concentrations
2.3 Heavy metal ions
2.4 Organic solvents
2.5 Surface active agents
 Physical Agents of Denaturation
Heat
• Gentle heating-reversible denaturation
• Vigorous heating disrupts several types of bonds
(H-bonds, ionic interactions, hydrophobic bonds)
• Destroys and coagulates protein in bacteria during
sterilization
• Heat coagulation is involved in cooking of foods,
testing of proteins in urine

Protein sample Heated HAc

White ppt. Precipitate
Egg albumin INCREASED
 Chemical Agents of Denaturation
1. Using conc. Mineral acids
• Disrupt H-bonds and salt linkages
• Conc. HNO3, HCl, H2SO4 and alkalies alter the
ionization of carboxyl and amino groups
• Protein will have excessive electrostatic charge of
one kind
– Polypeptide chain will unfold and seek an expanded
configuration to minimized repulsion
– Further addition redissolves proteins and hydrolysis
occurs later
• If acids or bases are contact with protein for a
long period of time, peptide bond will break
Protein sample + HNO3 + excess HNO3
White ppt. Precipitate disappeared
Egg albumin SOLUTION turned YELLOW
Chemical Agents of Denaturation

2. Using Salts of Heavy Metals

• Hg 2+, Fe 3+, Pb2+, Cu2+, Ag+ etc.
• Combine with carboxylate group forming
metal proteinates
• Coordination complex may also be form
between metal and free amine and other
groups in the proteins
• Disrupts salt bridges and disulfide bonds
• Poisonous if taken orally
(antidote:milk/egg white)
Chemical Agents of Denaturation
3. Using Alkaloidal reagents
 - biuret reagent is light blue but in the
presence of proteins it turned VIOLET
under alkaline conditions, peptide bonds are
cleaved and react with a copper reagent to yield
a purple color complex

• Compounds containing 2–CONH2 groups

O
joined either directly together or through O

a N or C atom react with alkaline HN

HN

copper sulfate giving violet- R H 2+

Cu
H R

colored coordination complex O

O
NH
NH
 - egg albumin when added with
- conc. HNO3  white ppt. is formed
- upon heating  sol’n. turns yellow
- when added NaOH  become ORANGE

• Involves the nitration of the phenyl group

upon heating with conc. HNO3forming yellow
nitro derivatives, and formation of orange
salts of these derivatives when made alkaline
with NaOH or NH4OH
– Positive reaction indicates presence of
tyrosine and/or tryptophan
– Phenylalanine is not readily nitrated and
does not or weakly responds to this test
- egg albumin when added
- with Millon’s reagent  white ppt. is formed upon
- adding NaNO2 and warmed  brick red ppt. formed.

• Due to the reaction of the hydroxyphenyl

group with the M-N reagent (HgSO4 in H2SO4)
forming a old rose-colored complexes which
is the mercury salt of the phenolic compound

– Any phenolic compound such as tyrosine,

phenol and thymol which is unsubstituted in
the 3,5 positions will be positive for the test
- egg albumin when added
- with Hopkin’s Cole reagent + H2SO4  a purple colored
liquid at the
junction of 2 liquids.

• The indole group in tryptophan condenses

with a number of aldehydes like glycoxylic acid
in the presence of conc. H2SO4= forming a
violet complex
 Indication of TEST
TEST/S POSITIVE for the
results presence of :

BIURET Violet Peptide

bonds
XANTHOPROTEIC Yellow (acidic) Tyrosine,
Tryptophan,
Orange (basic) aromatic
compounds
Tyrosine,
MILLON-NASSE Old rose Tryptophan,
Phenylalanine,
(pink) phenolic groups

HOPKINS-COLE Violet ring Tryptophan

 Experiment
ENZYMES & VITAMINS
Objectives
1. To be able to isolate and test the activity
of the enzyme catalase and ptyalin.
2. To be able to test and describe the
characteristic reactions of some water-
soluble and fat-soluble vitamins.
Definition of terms
Enzymes -

What are vitamins?

Vitamins
A. Enzyme Isolation and Tests catalase
Enzyme action: catalase – catalyze the decomposition
of hydrogen peroxide to
water and oxygen.
catalase
2 H2O2  2 H2O + O2
Oxygen
was liberated
hydrogen peroxide
Glowing splinter
continued to glow
Protein nature of Enzymes

filtrate + 10% NaOH + CuSO4 Positive to

Biuret test

colored solution
Starch + Iodine BLUE COLORED + Ptyalin
solution Starch-Iodine Complex

BLUE COLORED
Starch-Iodine Complex
Starch-Iodine Complex
+ more saliva Ptyalin + Iodine
POSITIVE
Solution to Benedict’s Test

Ptyalin is an enzyme called -amylase that catalyzed

the hydrolysis of starch . Saliva was the source of
ptyalin in this particular experiment. brick red
precipitate

starch left after hydrolysis by ptyalin but simple sugars

Ptyalin Ptyalin Ptyalin
Starch Dextrin
Salivary amylase (Ptyalin)
Components of saliva
– 99.49% water
– Mucin
– Mineral salt

Main function of saliva

1. Aid in swallowing the food
2. It contains enzyme that hydrolyzes starch to
dextrins and maltose and eventually GLUCOSE a
simple reducing sugar
B. Vitamins
Vitamin A - also known as RETINOL
Test : Carr-Price test
• Vitamin A + SbCl3 in CHCl3→ Blue

Carr-Price reaction is a test for

retinol. Retinol produces BLUE
COLOR in reaction with Antimony
chloride that fades after sometime.
Retinol is converted to anhydrous
retinol by transient thus blue color
fades.
Vitamin D - also known as SUNSHINE vitamin
• If Vitamin D is present, reddish brown precipitate
is observed that totally became solution

• The Vitamin D content of the

body can be increased by
exposure of the skin to
ultraviolet rays from the sun
but care must be taken to
avoid overexposure and
consequent sunburn.
Vitamin C
- as ASCORBIC ACID from fruits and are required for
the synthesis of collagen, epinephrine, proper
functions of bile acids, absorption of iron, and as an
antioxidant.
If Vitamin C is present,
precipitate will be observed that will
become solution
• The Vitamin C can be synthesized
by plants and most animals but
not by humans. A lack of this
vitamin produces a disease
known as SCURVY And also
causes a lack of intercellular
substance that cements the
tissues together.
Vitamin B1 - also known as THIAMINE that occurs
in yeast, milk, eggs, nuts, and whole
grains.
• If Vitamin B1 is present, a peach to pink to
yellow orange precipitate will form and
later will fade into light solution

• THIAMINE is necessary for the

normal metabolism of
carbohydrates. It also functions
in the utilization of pentose in the
hexose monophosphate shunt
and in some amino acid synthesis.

Thiamine Bismuth-KI
Vit. Common Source Defficiency
name
K anti-hemmorrhage green of
Control leafyHemorrhage
tissues Hemorrhage
vitamin

E anti-oxidant milk, eggs, fish, muscle meat,

Preservative Muscle destrophy to
(tocopherol) cereals, veg paralysis

B2 Riboflavin Yeast, milk, liver, heart

Metabolism of and leafy
CarbohydrateCheilitis (mouth
& lesions)
Fats
for proper functioning glossitis (tonue
vegetables of heart /nervous /digestion system
inflammation)
DNA repair
eggs,&milk
production of steroids, hormones
and fruits Pellagra or in adrenal
B3 Niacin glands
rough skin

B5 Pantothenic Liver, kidney, fish,and

Metabolism eggs, milk ofDegeneration
synthesis Carbo, in the adrenal
protein & fats
and lean meats cortex and impotency

B6 Pyridoxine Yeast, egg yolk, liver,

Balancing of Naand
& K grain
, promotes RBCdermatitis
production
germs
B7 Biotin Liver,
Cell kidney, ,fish,
growth eggs, milk & Metabolism
FA production
Dermatitis, muscle pains
of Fats & AA
and depression

B9 Folic Green leaves, yeast, liver, Megaloblastic anemia

kidney andof
Replication cauliflower gastrointestinal
DNA, maintenance /production of cells
disturbances

B12 Cobalamine Liver, kidney, person’s

Reduces fish, eggs, milk,risk ofPernicious
anemia anemia
oysters, and clams
 Experiment
CARBOHYDRATES
Objectives
1. To be able to perform tests that will classify
carbohydrates as reducing or non reducing
sugars.
2. To be able to observe the reactions of some
carbohydrates with Molisch test and iodine
test.
3. To be able to define the conditions necessary
in the hydrolysis of carbohydrates.
Carbohydrates substances that are polyhydroxyaldehyde or
polyhydroxyketones that yield them upon hydrolysis.

Sugars are CHO that are soluble in water and “taste sweet”
Monosaccharides  are the simplest sugar-CHO that can not be
hydrolyzed further
 are capable of reducing oxidizing agents like
KMnO4 or Br2 as a (“reducing” carbohydrates)
 Very solube in water, sparingly soluble in
ethanol, insoluble in ether
 Aldose, ketose (Tetroses, pentoses, hexoses)
D-glucose – known as dextrose or grape sugar
– Component of maltose, starch, dextrin, glycogen and
cellulose
– Blood sugar
D-fructose – known as levulose and fruit sugar
– Occurs in fruits and found in 1:1 ratio with glucose in
honey
– Component of table sugar (sucrose) and inulin
Oligosaccharides composed of 2-10 monosaccharides linked by
or Disaccharides acetal or ketal linkages and produces 2 mono-
saccharides upon hydrolysis
 Maltose (malt sugar) – intermediate in the hydrolysis of
starch to glucose
 Sucrose (cane or beet sugar) – occurs free in the plant kingdom
e.g. sugar cane, sugar maple, sugar beets
 Lactose (milk sugar) – found in milk, not very sweet, not
fermented by yeast
Polysaccharides are the more complex CHO
 made of monosaccharides, may also contain sugar
derivatives e.g. sugar acids or amino sugars
 structural function
cellulose, agar – agar
 nutrient function
starch, dextrin, glycogen, insulin
• Amylose Starch
 Not branched but helical in configuration
 Hollow core due to the α-(1-4) glycosidic linkage
• Amylopectin (80-85% of starch)
 Highly branched due to the presence of α-(1-6) glycosidic
linkage at every twelfth glucose that are joined by α-(1-4)
glycosidic linkages
 In presence of conc. H2SO4 glycosidic bonds
are hydrolyzed giving monosaccharides
which are then dehydrated to furfural,
hydroxymethylfurfural, etc.
These then react with α-napthol forming
purple-colored condensation products
Color at the
SAMPLE interface
OH H
REMARKS
O
H

H
H H
O
GLUCOSE
O O Violet +
H2SO4 H H H
+
H O
H FRUCTOSE H Violet
H O H O
H OH MALTOSE
H H Violet H H
+O
H H H
+
O
H H LACTOSE Violet
H H
SUCROSE Violet +
STARCH Violet +
COTTON Violet +
 Iodine test
• differentiates helical from non-helical
polysaccharides
Iodine is accommodated
Polysaccharides in hollow
Results core of
/ Observations
helix and complex is formed resulting to blue
1% Starch
color Blue-black solution
1% Agar-agar Brown yellow solution
Color differences are related to extent of
1% Gum Arabic Yellow solution brown)
coiling (e.g. purple> red> red-brown>

Inulin, gum arabic and agar-agar have linear

structures that are not coiled
Reducing Action of Carbohydrates
- test for the presence of aldehyde
Fehling’s test functional group but not the ketones
Two solutions were used in the test
Fehling's A – a blue solution which consists of
Cu2SO45H2O in distilled H2O + dil.H2SO4
Fehling's B – an alkaline colorless solution
containing 2,3-dihydroxybutanedioate
ions (tartrate ions)+ NaOH in dist.H2O

• Aldehydes are reducing agents,

• reduce the blue copper(II) ions to brick-red copper(I)
oxide, Cu2O, which forms a precipitate (suspension of small
particles produced in liquid by chemical reaction).

• Ketones are not reducing agents,

– no colour change is observed.
Benedict’s test
Uses reducing property of some sugars
Cupric ion is reduced and cuprous oxide
precipitates out of the alkaline solution
as brick red precipitate

Fehling’s test
• distinguishes reducing sugars, such as glucose,
which have a free aldehyde group, from non-
reducing sugars, such as sucrose (ordinary table
sugar)
• Reducing sugars form a brick-red precipitate
when tested
 Sample Benedict’s Fehling’s
test test
GLUCOSE +reducing+
FRUCTOSE +reducing+
MALTOSE +reducing+
LACTOSE +reducing+
SUCROSE Non-reducing
- -
STARCH Non-reducing
- -
COTTON Non-reducing
- -
Intensity of color will depend on the
amount of reducing sugar present (darker
for those with highest proportion of
reducing sugars)
Hydrolysis of Sucrose
On hydrolysis this disaccharide yields two monosaccharides

C12H22O11 Hydrolysis C6H12O6 + C6H12O6

sucrose glucose fructose
maltose glucose glucose
lactose glucose galactose

CHO reducing sugars


HO-C-H

HO-C-H

H-C-OH COOH
 
H-C-OH HO-C-H
 
HO-C-H
CH2OH 
H-C-OH
Glucose 
H-C-OH


CH2OH
Gluconic
Hydrolysis of Polysaccharide
On hydrolysis of starch it can also yield glucose upon completion

erythro
STARCH Hydrolysis DEXTRIN GLUCOSE
MALTOSE
Blue Reddish Colorless

Benedict’s test
Sample Unhydrolzed Hydrolyzed

SUCROSE - +
STARCH - +
1. Why does fructose, a ketose reduce Benedict’s reagent?
Fructose , base on its structure has an aldehyde
available for reduction reaction that is why
it was able to reduce Benedict’s reagent.
2. Why sucrose was negative to reduction test?
In sucrose (1,2 glycosidic structure), the glucose part
had the aldehyde at C-1 and the fructose part
had the ketone group at C-2. Since the linkage
is 1,2- neither is free thus sucrose is not
a reducing sugar
3. Why sucrose and starch become positive for Benedict’s
after hydrolysis?
Hydrolyzed sucrose become positive for Benedict’s
test because the product ; glucose and fructose
are reducing sugars
Also, hydrolyzed starch become positive too to
Benedict’s test because the end product of
complete starch hydrolysis is glucose
 Experiment
LIPIDS
Objectives
1. To be able to observe and describe the
properties and reactions of lipids.

2. To be able to prepare soap and describe its

properties.

3. To be able to isolate cholesterol from chicken

egg yolk or brain sample and describe their
chemical reactions.
 Lipids
Lipids- are the second group of organic
compounds that serve as food for the
body.
Lipids- a collection of organic molecules
united by solubility in nonpolar solvents.
Varying chemical composition
Three main group
(simple, complex and derived)
Uses of fats in our body
1. As fuel Fat: 9kcal/g
Carbohydrate: 4kcal/g
2. Reserve supply of food
– Stored in the adipose tissue
3. Protector of vital organ as shock absorber
– Surround the vital organ to keep them
in place
4. As heat and electrical insulator
– Keep our body warm and allow
propagation of nerve impulses
 Physical properties
Lipid samples
Properties Beef Butter Cottonseed
suet oil
Odor odorless odorless
unpleasant pleasant odorless
unpleasant

Appearance dirty
white yellow
yellow yellow
colorless
white
Physical soft
solid soft
solid liquid
liquid
state solid solid
Texture greasy
greasy greasy
greasy greasy
greasy
Solubility of coconut oil
Coconut oil is 44-51% Lauric acid
Lipids are nonpolar substances so they
are only soluble to nonpolar solvents

Solvent Solubility
Water immiscible / insoluble
IM
Cold ethanol immiscible / insoluble
Hot ethanol miscible / soluble
CHCl3 miscible / soluble
Ether miscible / soluble
M
Formation of • Volatility vs.
translucent spot translucent spot

Sample Result
Ethanol -
Ether -
Coconut oil +
Emulsification
Bile and soap are
Emulsification: making a colloidal
emulsifying suspension
agents
of one liquid with another liquid
Sample Results
Cotton seed oil +
H2O + soap Turbid mixture with tiny
solution droplets
Cotton seed oil + Milky mixture with tiny
bile solution droplets
Why is bile important in digestion of fats ?
 Bile contains bile acids which emulsify the lipids within
the water-insoluble droplets during the digestion.
Bile acids solubilize lipids by forming micelles which
act as emulsifying agents
Acrolein test
When a fat is heated strongly in the presence
of HSO4 (dehydrating agent) the glcerol portion
of the molecule is dehydrated to form the
unsaturated aldehyde called “ACROLEIN”
distinguished by a “BURNT OIL” odor.
H-C=O
• Chemical equation

H-C
Sample Results


H-C
CH2 OH 

Coconut oil
Δ / KHSO 4
2H2O +
+ H

O=CHCH=CH
+
2
H C OHGlycerol
ACROLEIN
Oleic acid -
CH2 OH
Saponification - soap are produced by
saponification
- heating of a fat with strong alkali or base will
form a soap

• Chemical equation

CH2OCO(CH2)10CH3
+ 3 KOH
CH2 OH
3 CH3(CH2)10COOK +
H C CHOCO(CH2)10CH3 Potassium oleate H C OH
a Soft or liquid soap
CH2OCO(CH2)10CH3 CH2 OH
Triolein
fats or oil Glycerol
Saponification
• Chemical equation

C17H35COOH + NaOH C17H35COONa + H2O

Stearic acid Sodium stearate water
a bar or hard soap

How to test if saponification is complete ?

Complete solubility of a drop of the solution
in distilled or tap water
 Formation of insoluble salt
• Chemical equation
CH3(CH2)10COO-K++ CaCl2 (CH3(CH2)10COO-)2Ca+2 + 2KCl
K soap Calcium soap
White precipitate
CH3(CH2)10COO-K++ MgCl2 (CH3(CH2)10COO-)2Mg+2 + 2KCl
K soap Magnesium soap
White precipitate
2CH3(CH2)10COONa+ (CH3(CH2)10COO-)2Ca2+ +2NaCl
+ CaCl2
Na soap Calcium soap
White precipitate
2CH3(CH2)10COONa++ MgCl (CH3(CH2)10COO-)2Ca2+ +2NaCl
2
Na soap Magnesium soap
White precipitate
Lieberman-Buchard Reaction
• Cholesterol gives deep blue green or emerald
green colored condensation products with
acetic anhydride and concentrated sulfuric acid
• Chemical equation
H3C

H3C CH3

conc. H2SO4
H3C CH3
O O
OH
+ CH 3 C O C CH3
Cholesterol Acetic acid
Salkowski test
•Chemical equation: 2 cholesterol → bisteroid
H3C

H3C CH3

H3C CH3

OH + conc. H SO
2 4

Cholesterol

Reddish brown
at the junction of
2 layered liquids