BIOCHEMISTRY

EXPERIMENT 1: ANALYSIS OF SUBCELLULAR COMPONENTS

➢ Sucrose solution isotonic (0.025M) was used to homogenate the chicken liver on the
experiment because it does not cross through the cell membranes and does not cause the
organelles to swell.
➢ Repeated centrifugation at progressively higher speeds will fractionate homogenates of cells
into their components. Smaller subcellular component = greater centrifugal force.
Homogenization- disrupting the cells under a mild condition using a blender.
Centrifugation- sub-fractionalization of the centrifugate.
Qualitative Tests- Biuret test (proteins), Sudan IV test (lipids), Molisch Test (carbohydrates),
Nucleic Acids (diphenyl colometric reaction), Orcinol Test (RNA), Dische Test (DNA)
➢ Nuclear Fraction (Nucleic Acids)- consists of nuclei (DNA and RNA) together with unbroken
cells.
➢ Mitochondrial Fraction (Carbohydrates)- consists predominantly of mitochondria.
➢ Microsomal Fraction (Lipids)- composed of the lighter cell organelles such as golgi bodies,
ribosomes, vesicles, and the cell membrane.
Pellet- the solid particle that settles at the bottom/ floats.
Supernate- the liquid part that comes out after centrifugation.

Why do the different macromolecules require different centrifugation sediment rates and times in order
to be isolated?
➢ Because particles of different densities or size will sediment at different rates with the largest
and most dense particles sedimenting to the fastest, followed by less dense and smaller
particles.
QUALITATIVE ANALYSIS
NUCLEIC ACID RNA
PROTEINS LIPIDS CARBOHYDRATES *DNA* (Orcinol/ Bial’s
(Biuret Test) (Sudan IV) (Molisch Test) (Dische Test) Tesr)

PELLET 1 ++ + + +++ +++

PELLET 2 + ++ +++ ++ ++

PELLET 3 +++ +++ ++ + +

 TEST REAGENTS COLOR CHANGE IMPORTANCE
α-naphthol dissolved Purple/ violet layer or A general test for
MOLISCH’S in ethanol, H2SO4 condensations; (+) to all carbohydrates
TEST
Cupric acetate w/ Brick red ppt of Cu2O Distinguishes
BARFOED’S HOAc Monosaccharide- fast; monosaccharides from
TEST Disaccharide- slow disaccharides
SELIWANOFF’S Recorcinol in HCl Fructose- red sol’n Test for ketone sugar
TEST Sucrose- brown ppt
OSAZONE TEST Phenylhydrazine in Yellow crystalline ppt, (+) Easily formed by
HCl, NaOAc to all glucose soluble reducing sugars and
used to identify sugars
FEHLING’S F1- CuSO4, Brick red ppt- Cu2O; Reducing sugar (sucrose
TEST Rochelle salts, KOH, sometimes yellow due to is negative)
NaOH Cu(OH)2, greenish due to
CuSO4
BENEDICT'S CuSO4CuNO3, Brick red ppt Test for reducing sugar
TEST Sodium Citrate
TOLLEN’S TEST AgNO2Ag (NH3)2 Silver Mirror (+) aldehyde group /
reducing sugar
IODINE TEST I and KI in ethanol Glucose - mahogany red Test for starch
Starch- Dark Blue
GALACTOSE
FRUCTOSE

MANNOSE
GLUCOSE

SUCROSE
MALTOSE

LACTOSE

STARCH
RIBOSE

SUGARS

TEST MONOSACCHARIDES DISACCHARIDES

MOLISCH’S + + + + + + + + +
BARFOED’S + + + + + + + + +
SELIWANOFF’S - + - - - - - + -
OSAZONE + + + + - + + - -
FEHLING’S + + + + + + + - -
BENEDICT’S + + + + + + + - -
TOLLEN’S + + + + + + + - -
IODINE - - - - - - - - +
BIAL’S - - - - + - - - -
 ➢ Ether, Alcohol- some are non-polar
➢ Ethyl and Methyl- polar
➢ Lipid derivatives- molecules originated from lipids (vitamins and hormones)
➢ Fatty acids- main component of the soap
➢ Simple- fats, oils, waxes, steroids
➢ Complex- phospholipids, sphingophospholipids, glycolipids
➢ Glycerophospholipids- esters of glycerol w/ fatty acids and phospholipid derivates

PRINCIPLES:
➢ Like dissolves like
➢ Solute dissolves best in solvent with similar chemical structure
➢ Solubility also depends on polarity
Saturated- single bonds (alkanes)
Unsaturated- double (alkenes) and triple (alkynes) bonded
➢ Functional group will break down the double/ triple bond and will be the point of attachment of
OH
Methane- simplest (CH4)

SPOT TEST FOR LIPIDS

➢ “grease spot test”
➢ Fats and oils have higher boiling points so at room temperature, they cannot absorb enough
heat to evaporate.
➢ Fat/oil defracts light when placed on a sheet of paper.
➢ Defracted light can pass from one side to another and produces a translucent spot.

IODINE TEST- test for unsaturation

➢ The more the double/ triple bonds are, the higher the degree of unsaturation.
➢ The Iodine sol’n will break the double/ triple bonds and attach to it.
➢ Unsaturated fatty acids- absorbs Iodine at the double bonds until all the double bonds are
saturated with Iodine.
Results:
Iodine + lipid = clear color (Unsaturated)
Iodine + lipid = brown color (Saturated)
Δ
ACROLEIN TEST- oil or fat glycerol + fatty acid
➢ To detect glycerol and fats
Dehydration- removing of water molecule
Acrolein- will produce pungent odor; formed due to the removal of water from glycerol.
KHSO4 (potassium bisulfate)- catalyst used in acrolein.
➢ When fat is treated strongly in the presence of dehydrating agent, the glycerol portion of the
molecule is dehydrated to form an unsaturated aldehyde.

SAPONIFICATION
(+) result- formation of bubbles
➢ Hydrolysis of an ester under basic sol’n to form alcohol and salts of COOH
➢ The head of the fatty acid is not soluble to dirt.
NaOH- most common basic solution; commonly used to refer to the reaction of metallic alkali
with a fat or oil to form soap. (Main ingredient of liquid sosa)
Emulsification- mixture of 2 liquids that are completely immiscible. (water + oil)
 Emulsifier- will make the immiscible sol’n mix together by breaking the barrier. (pinch of soap)
Lecithin- a natural emulsifying agent.

Amino Acids - are the basic unit of proteins.

• > 50 AA → proteins/polypeptides
• < 50 AA → peptides

R Polar Alcohol and amides

R Non - polar Alkene groups

R Acidic Carboxylic acids

R Basic Amides
*R- side chain

Peptide bonds - linkage between 2 amino acids

• Amino groups - carboxyl group
6.5 - 7 → Neutral Ph
Heat (Δ) - disrupts Hydrogen bonds and hydrophobic interaction between “R’ groups
• → production of precipitate, only the colors varies
→ White precipitate - sediment/ solid form
Heat (Δ) - destroys structures of proteins

Heavy Metals Salts - (+) charged will attached to the carboxylic group until the AA neutralizes
protein thus reaching the isoelectric point.

Results:
Lead Acetate white solution

Ferric Chloride yellow to brown solution

Copper Sulfate Blue solution

Strong Mineral Acids - mineral or salts/ alkaloidal agents are negatively charge but reacts with the
positive part of amino acids (amino group) and neutralize it thus reaching the isoelectric point.

Results:

Tannic Acid forms white to light brown ppt

Citric Acid yellow floating ppt

Strong Acids
Principle: splits salt linkages by ionizing the COOH group.
Results:
Conc. Nitric Acid cloudy white precipitate

Conc. Sulfuric Acid brown sol’n white precipitate

Alcohol
Denaturation - changes in the structure of the protein
Principle: denatures proteins by disrupting the hydrogen
Result:
Alcohol white sol’n with white precipitate

Biuret Test - test for all proteins

Principle: detection of peptide linkages
Results:

Reagent

Copper Sulfate and NaOH Blue Sol’n

Millon’s Test - detection of phenol derived AA (Tyrosine)

Results: (Note: positive occurs when tyrosine is present)
Reagents

Mercury in Nitric Acid Red/ Flesh Sol’n

Hopkin’s Cole Test - specific test for tryptophan (indole - containing AA)
Principle: reaction with glyoxylic and with strong acids.
Results:

Reagents

Glyoxylic Acid + Sulfuric Acid Purple/ Violet Sol’n

Molisch Test - test for carbohydrates

Principle: dehydration of the carbohydrates by sulfur and hydrochloric acid to produce an aldehyde (-
CHO)
Result: (Positive for glycoproteins)
Reagent

Alpha-naphthol in alcoholic sol’n + sulfuric acid Purple/ Violet ring

Reduced Sulfur Test

Principle: detection of AA containing SH or S (Methionine, Cystine, Cysteine)
Results:
Reagents

Lead Acetate and alkaline sol’n (KOH, NaOH) Black sol’n or deposits
ACIDITY AND BASICITY
AMINO ACID ACID, NEUTRAL, BASE

Glycine N

Arginine B

Histidine B

Alanine N

Lysine B

Aspartic Acid A

EXPERIMENT 5: AMINO ACIDS

Amino Acids - amine + carboxylic acid
Classified according to the relative position of the final group of the following:
• Gaba - most commonly known gamma AA
• Glycine - simplest amino acid

Non - polar CH3, CH2

Polar CONH2, OH

Acidic COOH

Basic N

Non- protein side chains

• Hydrophobic R are chemically unreactive
• tend to aggregate rather than be exposed to the aqueous environment
• tend to be found on the interior of proteins
• water hating
Polar Side chains
• involves hydrogen bonding interactions
• cysteine is important because its ability to form disulfide bonds
• proteins become negatively charged because of the acidic side chain

Small Glycine, Alanine

Branched Valine, Leucine, Isoleucine

Hydroxy (-OH group), Serine, Threonine

Sulfur Cystine, Methionine

Aromatic Phenylalanine, Tyrosine, Tryptophan

Acidic and their derivatives Aspartate, Asparagine, Glutamate, Glutamine

Basic amino Lysine, Arginine, Histidine

Imino Proline
Alanine - longest distance AA that traveled (paper chromatography)
Aspartic & Histidine - shortest distance
Ninhydrin - generation for amino that will yield to purple/violet color

➢ Most enzymes are globular proteins (almost all of enzymes are proteins except enzymes that
are chemically prepared).
➢ Factors affecting enzyme reaction: temperature, pH, substrate concentration, enzyme
concentration

RESULTS:
OXIDASES
➢ Enzyme discoloration
➢ Produce benzoquinones and melanins (caused by phenolases)
Benzoquinones- Brown discoloration
Polyphenol oxidase- initiates discoloration
Phenols- substrate that acts upon the enzyme
Phenolases- enzyme that acts upon the substrate to produce brown discoloration

RESULTS:
SAMPLE USED TIME OXIDIZED
Apple 2 minutes
Banana 15 minutes
Potato 16 minutes
Guava 32 minutes
*Guava contains Vitamin C which is an antioxidant, preventing the phenolases (enzyme) from
discolorating it.

PEROXIDASE
➢ An enzyme that decomposes H2O2 with Guiac solution
Potato extract- contains substances that can be oxidized
Potato peroxidase- decomposes H2O2 and organic peroxidases to produce oxygen

RESULTS:

TEST TUBE 1 Extract + Phenol + H2O2 Brown solution

Extract + Catechol/ Pyrogallol +

TEST TUBE 2 H2O2 Brown solution
Extract + Guiac solution +
TEST TUBE 3 H2O2 Blue solution
BOILED EXTRACT No change

ANIMAL OXIDASE
Paraffin oil- prevents reoxidation of Methylene blue
Formaldehyde (HCHO)
▪ Substrate
▪ Reduction of Methylene blue
 Methylene Blue
▪ Indicator (H acceptor)
RESULTS:
TEST TUBE 1 Fresh milk + MB + HCHO + Intense blue
Paraffin oil + heat again
TEST TUBE 2 Fresh milk + MB + HCHO + Lightest blue solution
Paraffin oil
TEST TUBE 3 Fresh milk + MB + Paraffin oil Lighter blue coloration than
Test Tube 1

Test tube 1- heat denaturated the enzymes present in milk. It killed most of the organisms found in
milk. Denaturation prevents the enzymes from catalyzing the reaction.
Test tube 2- great amount of Methylene Blue is reduced
Test tube 3- no HCHO was added (scarcity of substrate). Few enzymes will be able to catalyze the
reaction

DECOMPOSITION OF H2O2 BY CATALASE

Catalase- promotes the conversion of hydrogen peroxide to water and oxygen
➢ Heme- containing proteins

catalase
H2O2 H2O + O

RESULTS:

TEST TUBE 1 H2O2 No color

TEST TUBE 2 H2O2 + liver (mongo sized) Bubble formation/
effervescence
TEST TUBE 3 H2O2 +MnO2 Bubble formation/
effervescence

➢ Bubble formation or effervescence- indication of liver’s action of breaking down Hydrogen

peroxide to water and oxygen.
➢ MnO2 (Manganese oxide)- chemically prepared catalyst/ inorganic

TEST FOR GENUINE HONEY

➢ Iodine solution- used to detect starch

➢ If the honey is not genuine, the solution will turn blue/ blackish (contains starch).
➢ If the honey is genuine, the solution will remain yellow.