EXPERIMENT 7A

ISOLATION, HYDROLYSIS, AND QUALITATIVE ANALYSIS OF RNA FROM YEAST

NUCLEIC ACIDS
● Macrobiopolymers of high molecular weight with mononucleotide as the repeating unit
● Information molecules
● The two structural kinds of nucleic acids are DNA and RNA, which are basically made
up of nitrogenous bases, sugar, and phosphate group
● Tissues with
○ High nuclear/cytoplasmic ratio,
○ High DNA concentration;
○ With high cytoplasmic volume,
○ High RNA value
● Major components of all cells (5-15% of their dry weight)

THREE TYPES OF INTERACTIONS (responsible for the rigid molecular configuration

of nucleic acids)
● Phosphodiester bonds- join the nucleotides in each chain
● Hydrogen bonds- join the bases
● Van der Waal’s fores- between stacked baes
(Isolation should be conducted such that drastic changes in their structures are avoided or
minimized)
GENERAL PROCEDURES FOR ISOLATION OF NUCLEIC ACIDS
● Disruption of cell membrane and membrane of subcellular particles to release the
nucleic acids
● Treatment with a solution which that will cause dissociation of the nucleoprotein and
denaturation of proteins
● Purification of nucleic acids

SACCHAROMYCES CEREVISIAE (YEAST)

● Unicellular fungus
● Contains 4% RNA by weight

ISOLATION OF YEAST RNA

● Heating with alkali (NaOH): extracts nucleic acids and water soluble proteins and
inactivates nucleases which can degrade RNA
● The nucleic acid is then separated from associated protein and other interfering
substances by acid extraction at pH 4-5
● Treatment with alcohol and conc. HCl: can precipitate the RNA
● Repeated washings with alcohol and ether/organic solvents: can remove lipids

HYDROLYSIS OF NUCLEIC ACID BY EITHER CHEMICAL OR ENZYMATIC

METHODS, YIELDS:
● Purine Bases (adenine and guanine)
● Pyrimidine Bases (cytosine, thymine, uracil)
● Oligonucleotide containing up to 20 residues
● Nucleosides (base + sugar)
● Ribose or D-Ribose
● Phosphates
Pyrimidine are more resistant to acid hydrolysis
Hydrolysis: H2SO4 and Heat
Purine-N-glycosyl bond is susceptible to acid hydrolysis

Nucleotides (phosphates + base + sugar)

 EXPERIMENT 7B
QUALITATIVE TESTS

Result from Samples

Name of Test Definition
Hydrolysate Unhydrolyzed RNA

Benedict’s Test for reducing sugars Brick-red precipitate Blue solution (no
Test visible reaction)
Aldehydes and ketones are oxidized by
alkaline copper solution. The cupric ions
are reduced to cuprous and brick red Cu2O
forms

Orcinol Test Test for pentoses Blue green solution Blue green solution

Decomposition of pentoses when heated

with concentrated HCl to form furfural
which then condenses with orcinol (3,5-
dihydrotoluene) to form a blue colored
compounds

Test for Hydrolysate + NH4 + AgNO3 → whitish Whitish precipitate Grayish-white

Purine Bases precipitate/ flocculent ppt precipitate

Unhydrolyzed + NH4 + AgNO3 →

grayish-white precipitate
Test for Clear solution with Yellow solution with
Inorganic yellow precipitate yellow precipitate
Phosphate

EXPERIMENT 8

Alimentary digestion
- The process whereby complex biomolecules present in food are broken down
into simpler substances useful to the body
- Requires presence of the digestive enzymes

- Breakdown of naturally occurring foodstuffs into assimilable form is the work of

hydrolytic enzymes, enzymes involved in digestion
● These enzymes catalyze the hydrolysis of proteins to amino acids,
starches to monosaccharides, and fats to acyl glycerols
Saliva
- A watery secretion produced by three pairs of large salivary glands plus
numerous minute glands, the buccal glands, which are located in the mucosal
lining of the mouth
- Speeds up chemical changes in the carbohydrates of ingested food materials
- It has no chemical effect on proteins and fats

Saliva contains:

1. 99% of saliva is water

2. Salivary amylase/ptyalin - a digestive enzyme which hydrolyses starch to
maltose
3. Mineral salts (e.g., sodium hydrogen carbonate) - helps maintain a pH of around
6.5 - 7.5 which is the optimum for the action of salivary amylase
4. Mucin - a sticky material (glycoprotein) which helps bind food particles together
and lubricate them to assist swallowing

Approximate pH of saliva with a pH paper: 8.0

● Saliva has a pH between 6.5 and 7.5

● Saliva is slightly alkaline, about neutral, owing to the presence of sodium
bicarbonate
● Salivary amylase functions best at the pH range

QUALITATIVE TESTS

Test Definition

Test for Mucin A sticky material which helps bind food

particles together and lubricate them to
assist swallowing

Heavily glycosylated protein found

extensively in salivary secretions

Can be precipitated out of saliva by

adding dilute acetic acid

Increase the viscosity of the fluids in

saliva (because of the highly extended
and highly hydrated structure)
Test for Proteins (Biuret Test) Gives a positive result if a violet-colored
solution is produced

Biuret complex: a compound containing

two - CONH2 joined either directly
together of through a N or C atom

If test sample is reacted with alkaline

CuSO4 and if it contains proteins in it,
then a violet color coordination
complex is produced

The intensity of the color is dependent on

the number of the peptide bonds present

Test for Inorganic Matter Test for Chlorides

Color of ppt: white

Name of ppt: Silver chloride (AgCl)
Equation: Ag+ + Cl- —> AgCl

Chloride reacts with AgNO3 to yield a

white ppt

Test for Phosphates

Color of ppt: yellow

Name of ppt: Ammonium
phosphomolybdate (NH4)3PO4(MoO3)12
Equation: PO43- + 3NH 4 + (aq) + 12 MoO2-
(aq) + 24 H+ + (NH4)3PO4(MoO3)12 + 12
H2O

Phosphate residues of phospholipids will

react with ammonium molybdate,
(NH4)2MoO4, in HNO3; a yellow ppt is
formed

Test for Sulfates

Color of ppt: white

Name of ppt: Barium Sulfate (BaSo4)
Equation: Ba2+ + SO42- —> BaSO4

Sulfate reacts with BaCl2 to yield a white

colored ppt
 Test for Calcium

Color of ppt: white

Name of ppt: Calcium oxalate (CaC2O4)
Equation: Ca2+ + C2O42- —> CaC2O4

Calcium reacts with (NH4)2C2O4 to yield

a white ppt

Calcium helps maintain or stabilize a

compact conformation of the molecules of
a-amylase and also helps maintain an
enzymatically active conformation

Test for Thiocyanate (FeCl3/Ferric

chloride test) Color: pale yellow sol’n with red ppt

Equation: 2 Fe3+ + 12 CNS → 2

Fe(CNS)63-

Thiocyanate reacts with FeCl3 in acidic

medium to yield a red-colored product

Thiocyanate is the metabolite of cyanide

and the end product of the detoxification
compounds containing cyanide through a
reaction catalyzed by the enzyme
rhodanase

It was proven that everyone has

thiocyanates in their saliva (from
smoking and foods containing
cyanide)

Digestion of Starch Paste

Description Reason from the Discussion

Starch forms a violet-colored complex - Iodine (I2) is used to detect the presence
with I2 of starch.
- Dark Violet complex means that the
starch has a long peptide.

This is possible because of the helical - Amylose is linear and not branched.
 form of a molecule of amylose or - Amylopectin is highly branched.
amylopectin.

When starch is digested by saliva, it is - The starch is digested, which means

broken down into smaller saccharide that the peptide is cut smaller and
units. It can no longer form the smaller. This causes the change of color.
violet-colored product when salivary
digestion is completed.

Summary:

↑ minute = ↑ more fainting of violet-colored complex

Note: The longer the starch is in the saliva, then the fainter it is when added by the I2 because it
is an indication that the starch is slowly being digested (means a shorter peptide).

Achromic Point

● Breakdown of starch is complete and starch has been converted to dextrin and
smaller monosaccharides and disaccharides.
● This is a point at which the reaction mixture no longer gives a color change with
I2 solution.
● The activity of the salivary amylase is enhanced by the chloride ion which is
present in saliva.
○ Salivary amylase - primary enzyme in saliva. Breaks down carbohydrates
into smaller molecules, like sugar.

Salivary Amylase

● Cleaves or split ⍺-1,4 linkages in starch

● Gives different colors in I2:
1. Amylodextrin = purple
2. Erythrodextrin = red
Dextrin = yellow
3. Achroodextrin = colorless
4. Maltose = colorless
 Digestion of Starch Paste
Diagram

Starch (black or dark in color) → Amylodextrin (purple) → Erythrodextrin (red) →

Dextrin (yellow) → Achroodextrin (colorless) → Maltose (colorless)

Test Description Positive Result Equation

Fehling’s Test Detects the Blue solution with

presence of brick-red precipitate
reducing sugars.

Phenylhydrazine The Crystals

Reaction monosaccharides
produced after
salivary digestion of
starch will react
with
phenylhydrazine
reagent to produce
Osazone crystals.

Benedict’s Test Test for reducing Green

sugars. Yellow
Red precipitate
The aldehydes and All sugars
ketones are
oxidized by alkaline Negative:
copper solution. Sucrose
The cupric ions are
reduced to cuprous
and brick-red Cu2O
forms.
 Influence Description pH Result

Influence of Free Free acid hinders Acid ↑ conc. of dil. HCl

Acid the digestion of Low level =
starch by salivary ↑ Intensity of blue
amylase. color

The more dilute the

acid, salivary
digestion will
proceed.

Influence of Alkali Same trend is Basic ↑ conc. of dil.

observed when High level Na2CO3
iodine test is done =
with additional ↑ Intensity of blue
dilute base. color

The more dilute the

base, salivary
digestion will
proceed.

Summary:

The saliva must remain pH 6.5 – 7.5 to completely break down starch and change into
lighter colors.

Not too acidic and not too basic. Only 6.5 – 7.5 !