EXP 1A: DIFFERENTIAL CENTRIFUGATION EXP 2B: TESTS

- Biomolecules cluster together to form more complex structures DISCHE/DIPHENYLAMINE TEST

Cell membrane: lipid/protein Cytoskeleton: fibrous protein structure - for DNA/deoxyribose in DNA
Chromatin: DNA/protein Virus- assemblage of DNA/RNA strand - Reagent: diphenylamine solution (diphenyl., glacial acetic acid, sulfuric acid)
Ribosome: RNA/protein wrapped in protein package - Positive: blue solution

BIURET TEST
- proteins
- Reagent: KOH/NaOH, hydrated CuSO4; Potassium sodium tartrate
- Positive: Violet solution

Step 1: Mechanical Disruption MOLISCH TEST

1. Protein purification- disrupt tissues & cell in controlled fashion - carbohydrates
2. Homogenization- rupture plasma membranes to release cell contents - Reagent: α-naphthol in ethanol, Sulfuric Acid
- organelles must still be intact in its membrane - Positive: purple ring in junction of 2 layers
3. Homogenate/Extract- has cytosol molecules (enzyme, ribosome,
metabolites) & membrane-enclosed organelles
Sucrose- homogenizer; contains macromolecules inside organelles
- maintain isotonic env to prevent movement of molecules
SUDAN TEST
- lipids
- Reagent: Sudan IV (red-brown dye nonpolar, soluble only to lipids)
- Positive: 2 layers (red-orange layer on top w/ red globules)
- no chemical reaction—only solubility test
- dye adsorb (stick on another substance’s surface) to lipids

THEORETICAL RESULTS

Step 2: Differential Centrifugation

- repeated centrifugation at progressively higher speeds to fractionate cell
homogenates into components based on size & density
- more dense: greatest centrifugal force & move most rapidly (bottom)
- less dense: supernatant (suspension on top)
1st Cent: Pellet 1 (unbroken cells, nuclei)
2nd “ : Pellet 2 (mitochondria, lysosome, peroxisome)
3rd “ : Pellet 3 (microsome, small vesicles)
Supernatant 3 (proteins, inorganic ions) EXPERIMENTAL RESULTS
 EXP 2A: PROTEINS BIURET TEST
Aspartame- sugar, dipeptide; carboxylic acid & methyl ester - general protein test; detect peptide bonds
Reagent: KOH; hydrated CuSO4 (crucial comp), potassium sodium
Positive: violet solution
- blue color of Cu2’s basic solution turns violet (> tripeptide is present)
- NaOH raises solution’s pH to alkaline levels

NINHYDRIN TEST
Glutathione- tripeptide (cysteine, glutamic acid, glycine) - general test for proteins (except proline:yellow)
- antioxidant & detoxifying agent - for -NH2 group in free amino acid
Egg Albumen Reagent: Ninhydrin Positive: deep blue/purple solution
- Ninhydrin degrades AA into aldehydes, ammonia, & CO2 (pH 4-8) then
condenses w/ ammonia & hydrindantin to produce Ruhemann’s purple

HOPKINS-COLE TEST
- Tryptophan indole group
Reagent: Hopkins Cole reagent & conc. H2SO4
Positive: purple ring at interface of 2 liquids
- dehydration of tryptophan w/ indole group reacts w/glyoxylic acid using H 2SO4
SAKAGUCHI TEST
- guanidine group (arginine)
Reagent: α-Naphthol, drop of sodium hypobromite
Positive: red complex solution
- arginine reacts w/ α-Naphthol & sodium hypobromite (oxidizing agent)

XANTHOPROTEIC TEST
Evaporated Milk - activated aromatic rings (tyrosine & tryptophan)
Reagent: conc. nitric acid, ammonium hydroxide
Positive: yellow solution/precipitate
- tyrosine & tryptophan have activated benzene ring & readily undergo nitration;
phenylalanine has benzene ring but inactivated
- heating w/ nitric acid gives color (tryptophan: orange; tyrosine: yellow)
- adding 50% sodium hydroxide (strong base) deepens color to orange

EXP 2B: PROTEIN DENATURATION

Denaturation
- conformation in modification not accompanied by peptide b. rupture
- secondary: disrupt hydrogen bonds
- complete structure loss produce unstructured protein strand
- extensive denaturation changes are irreversible
Effects: decreased solubility Improved digestibility
Altered water binding capacity Increased intrinsic viscosity
Loss of biological activity Inability to crystallize
Toxin destruction
Renaturation- limited denaturation changes can be reversed (“refolded”)
STRONG ACID
- acid & base disrupt salt bridges held by ionic charges
- HNO3 (yellow), H2SO4 (white)
- AA residue protonation changes whether or not they participate in
H bonding, so change in pH denatures protein
- Salt bridges result from neutralization of acid & amine on side chains;
interaction is ionic between (+) amino group & (-) acid group

HEAT
- disrupt H bonds & nonpolar hydrophobic interactions
- increase KE, causing molecules to vibrate rapidly & violently
- medical supplies are sterilized by heating to denature bacteria proteins
- high level of thermal energy disrupt H bond; unfold & lose function

ALKALOIDAL REAGENTS
ALCOHOL - high molecular weight anions; combine w/ amino group to disrupt ionic bond
- H bonding between amide groups in secondary structure - (-) charge of anion counteracts w/ (+) charge of amino, forming precipitate
- H bonding between "side chains" in tertiary structure Picric Acid: Yellow coagulate Trichloroacetic acid: milky white c.
- disrupt side chain intramolecular H bonding by interacting w/ H bond Tannic Acid: Flesh “
- new H bonds are formed instead between new alcohol molecule & side chains
- 70% alcohol (skin disinfectant) since it penetrates & denature bacterial cell wall
- 95& alcohol merely coagulates protein on outside cell wall & prevents any alcohol
from entering cell

HEAVY METALS
- ions form strong bonds w/ carboxylate anions of acidic amino acid/cysteine 3A: ENZYMES
SH groups, disrupting ionic & disulfide linkages PEPSIN
- heavy metal salt reacts w/ protein, forming insoluble metal protein - hydrolyze
salt protein to peptide to amino acids
target: PEPTIDE BOND between hydrophobic & aromatic AA
- disrupt disulfide bonds due to high affinity & attraction for sulfur
optimum pH: 1.5-2.0; substrate: ALBUMIN
- mercury & lead interact w/functional side chain groups to form complexes
- oxidize amino acid chains Peptide pepsinogen- released by chief cells in stomach wall; activated by
hydrochloric acid of gastric juice, becoming pepsin
- Hg2+, Pb2+, Cu2+ are high molecular weight cations
- (+) cations counteracts w/ (-) of carboxylate group, forming precipitate
 Yellow = ENZYMES STILL PRESENT

* Buffer should be used instead of acid since acid hydrolyzes starch; sodium
AMYLASE carbonate reacts w/iodine, forming colorless solution
- breaks starch to sugar (MALTOSE: end product of s. amylase catalysis) Experimental: B1 (acidic)- salivary amylase is not denatured (dark yellow)
Glucosemaltosedextrinamylosestarch B2 (basic) – colorless (most digestion, but UNDETERMINED)
B3 (buffer)- least digestion=low amount of hydrolyzed starch (blue-gray)
Salivary amylase/Ptyalin
- saliva; optimum pH: 6.7-7.0; substrate: STARCH ENZYME CONCENTRATION
- target: α-1, 4-glycosidic bonds in starch, producing oligosaccharides
- when enzyme is no longer available, it reaches maximum point
(maltose/glucose) depending on time spent in mouth
- when conc is continuously increasing, reaction rate still increases
Iodine Test- reacts w/starch, forming blue-black solution
- enzyme dictates reaction rate
- NEGA = POS DIGESTION/HYDROLYSIS (vice-versa)

SUBSTRATE CONCENTRATION
CATALASE
- doesn’t affect enzyme activity as long as enzyme amount is sufficient
- oxidase; protects organisms from hydrogen peroxide effects - velocity increase = substrate conc increase
- H2O2 (powerful oxidizing agent; potentially damaging to cells) - when enzyme is no longer available, Vmax is reached (saturation point)
- substrate: HYDROGEN PEROXIDE; products: OXYGEN & WATER
- optimum pH: 7-11

Potato catalase- optimum pH: 9.0

BUBBLES- indicator that catalase already acted on substrate

Activation Energy- initial energy input

3B: FACTORS AFFECTING ENYZME ACTIVITY
TEMPERATURE- increase enzyme activity up to optimum temp

Mechanism
- Biological systems are very sensitive to temp changes.
- enzymes increase reaction rate of reactions w/o temp increase by lowering
activation energy (new reaction pathway)

Cold temp slows down enzyme activity

Boiling potatoes totally denatures catalase

pH- depends on enzyme’s optimum; extreme levels cause denaturation
- salivary amylase: optimum pH 6.8
4A: DNA EXTRACTION
Homogenization solution: sodium chloride, hot water, dishwashing liquid
BLENDING- crush cell wall
DISHWASHING L.- disrupt membrane proteins & lipid bilayers
- push DNA to salt solution
As dissolved in solution, it needs to precipitate & solidify for collection
IODINE TEST- presence of starch
SODIUM CHLORIDE- increase DNA separation from hydrophobic layer
Bluish-black = DENATURATION/HYDROLYSIS - DNA repel each other & do not allow grouping, aggregating it
Water molecules around DNA makes it difficult for salt ions to interact w/ DNA
ETHYL ALCOHOL-repel water from DNA; for aggregation
 - since its polar; it attracts salt (+) & wash it away from DNA
- as DNA is insoluble to ethanol, it precipitates
 DNA molecules aggregate & appear as cloudy, mucus-like strings BIAL’S/ORCINOL TEST
- pentose (ribose); NEGATIVE for DNA
DIPHENYLAMINE TEST (NOT ALL NUCLEIC ACIDS ARE POSITIVE IN TEST)
reagent: orcinol, hydrochloric acid, ferric chloride (FeCl 3)
Reagent: sulfuric acid, glacial acetic acid, diphenylamine positive: blue/green solution (hydrolyzed)
(-)
negative:
(-) principle: In acidic env, pentose forms furfural which condenses w/ orcinol in
presence of Fe3, producing blue/green solution
(+)

(+)

EXP 4B: DNA HYDROLYSIS TEST FOR PHOSPHATE

- phosphate ions; for total hydrolysis, phosphate must be separated from sugar
ACID HYDROLYSIS (Lysis- breakdown)
Reagent: ammonium hydroxide, nitric acid, ammonium molybdate
- strong acidic solution breaks down nucleotides into
Positive: bright yellow solution & precipitate (total hydrolysis)
- done by nucleases that cleaves phosphate diester bond
- deoxyribose & phosphate still attached to each other (partial hydrolysis) Negative:
Principle: Acidifying sample using nitric acid produces hydrogen phosphate, then
detected by ammonium molybdate, forming yellow precipitate of
ammonium phosphomolybdate

FEHLING’S TEST
- reducing sugar (monosaccharides: glucose, fructose, ribose)
- if ribose is separated/hydrolyzed from DNA chain (from base/phosphate)
- sugar must be separated before giving positive result
Reagent: F. A: copper sulfate
F. B: aqueous potassium sodium tartrate, sodium hydroxide
Positive: brick red precipitate (hydrolyzed)
Protonation Negative: dark blue (unhydrolyzed)
- donates H+ to base’s N7 to start reaction (causing depurination by hydrolysis) Principle: Heating reducing sugars w/reagent develops red-brown precipitate
 use water to to break nucleic acids to nucleotides
 further protonation: purine bases are separated
 further: separate phosphate+sugar
Depurination
- breakdown β-N-glycosidic bond, releasing adenine/guanine (if pH is 3)
- products: purine base & deoxyribose+phosphate group
Total hydrolysis: breakdown glycosidic bond2/phosphodiester bond if pH < 2
extremely low pH=complete DNA hydrolysis
products: phosphate group, purine/pyrimidine, deoxyribose

DISCHE TEST
- hydrolyzed & unhydrolyzed are both POSITIVE as it tests DNA chain
(hydrolyzed): bright blue; (unhydrolyzed): brownish blue
- doesn’t confirm if hydrolysis happened

TEST FOR PURINE BASES

EXP 5A: LIPIDS
- Adenine & Guanine; test for DEEP PURINATION, not hydrolysis Samples: OLIVE OIL- predominantly w/oleic acid
Reagent: silver nitrate & ammonium hydroxide COCONUT OIL- “ “ /lauric acid
LECITHIN- phosphoglycerides w/aminoalcoholcholine
Positive: white gelatinous precipitate (hydrolyzed) GLYCEROL/GLYCERINE
Negative: brown solution (unhydrolyzed)
TRANSLUCENT SPOT TEST
Principle: Ag+ in ammoniacal solution precipitates purines, reacting to N of purine
- preliminary; not confirmatory; lipid-like (not necessarily lipids)
- not all positive are lipids; all lipids are positive
Principle: lipid will not wet filter paper, forming a greasy spot (translucent spot)
UNSATURATION TEST
unsaturated fatty acids/carbon-carbon double bonds
Positive: pink disappear to clear by adding unsaturated FA EXP 6: CARBOHYDRATES
Negative: pink will not disappear BENEDICT’S TEST
Principle: all neutral fat has glycerides of fatty acids. Double bond in unsaturated FA - reducing sugars; copper oxide: responsible for color
becomes saturated by taking up bromine/ iodine. If lipid contains more
Reagent: Copper sulfate, Sodium carbonate, Sodium citrate
unsaturated FA, it will take more iodine
Positive: (+) Green, slight yellow precipitate (trace) (++) Green, thick yellow ppt (1g/100mL)
ACROLEIN TEST (+++) Yellow, orange ppt, (2g/100mL) (++++) Orange, orange to red ppt (> 2g)
- glycerol/fat Negative: blue
Reagent: potassium bisulphate (KHSO4) (dehydrating agent) Principle: Copper sulfate reacts w/free ketone/ aldehyde of reducing sugar to form
Positive: pungent smell w/slightly black solution (acrolein polymerization) yellowish orange/red ppt
Negative: no pungent smell (glycerol isn’t dehydrated/no glycerol) BARFOED’S TEST
Principle: As lipids w/glycerol w/reagent are heated, glycerol is dehydrated to form
- aldose/ketose monosaccharides; copper (I) oxide: responsible for color
acrolein. Further heating results to acrolein polymerization
- disaccharides undergo same reaction but slower
SOLUBILITY TEST Reagent: copper sulfate & acetic acid Positive: brick red precipitate
- preliminary; solubility of all lipids in solvents whether it’s miscible/immiscible Principle: Monos are oxidized by copper ions, forming carboxylic acid & brick red
Positive: lipids soluble in nonpolar solvents (chloroform); partially soluble in ethanol precipitates of copper (I) oxide
Negative: “ insoluble in polar solvent (water) (layering) SELIWANOFF’S TEST
Principle: Lipids are readily miscible in non-polar solvents (chloroform), partially - ketoses; dehydrated ketose react faster than aldohexose
soluble in polar solvent (ethanol), immiscible in polar solvent (water)
Reagent: resorcinol & hydrochloric acid
Positive: bright cherry red (ketohexose: fructose & sucrose); yellow-pale pink (aldohexose)
Principle: reagent dehydrates ketohexose to form 5-hydroxymethyl furfural, which further
reacts with resorcinol
EXP 5B: ANALYSIS OF LIPIDS BIAL’S/ORCINOL TEST
Lipid Extraction - pentose
- lipid characterization depend on its solubility in organic solvents, immiscibility Reagent: orcinol, hydrochloric acid, ferric chloride
w/water, physical characteristics (relatively low density) Positive: bluish-green (pentose) Negative: yellow-brown (hexose)
- Principle depends on lipid polarity compared to solvent polarity Principle: pentose forms furfural which condenses w/ orcinol in presence of ferric ions
- Polar (glycolipids, phospholipids): more soluble in polar solvents (ethanol/acetone), MUCIC ACID
- non-polar (triacylglycerols): more soluble in nonpolar solvents (hexane, cyclohexane) - galactose
- lipids vary in polarities & unlikely to select single organic solvent to extract them all Reagent: concentrated nitric acid

Yolk Composition Positive: mucic acid crystal formation (absence means that sol’n can still have carbo)
- mixture of lipoproteins (16% protein; 32-35%, lipids) Principle: aldose+acid forms dicarboxylic/mucic acid (insoluble in water)
- lipid fraction: 66% triglycerides, 28% phospholipid, 5% cholesterol, other lipids MOORE’S TEST
- Cholesterol is found only in yolk; concentrated w/lipids (1/3: 2/3 neutral, 1/3 polar) - reducing sugars (except sucrose)
- neutral lipids: sterolester, carotenoid, triglyceride, fatty acid, diglyceride, sterol Reagent: concentrated sodium hydroxide
triglyceride is predominant Positive: caramel odor & yellow/orange/dark brown solution (galactose, glucose, maltose,
- Position 1 of triglyceride is mostly occupied by palmitic acid (saturated acid) Negative: glycogen, starch, sucrose fructose, lactose)
- Position 2 by oleic & linoleic acids (unsaturated acids); Principle: Aldol condensation (caramelization reaction) under alkaline/basic conditions in
- contain Lecithin (glycerophospholipid, w/atty acids, glycerol, phosphate, choline) presence of free carbonyl groups of reducing sugar
Solvent: T1: Ethanol (polar; w/glycolipids) (Molisch)
T2: Cyclohexane (nonpolar; triglyceride, choles) (Ninhydrin, Acrolein, Salkowski)
T3: Acetone (slightly polar; w/phospholipid) (Acrolein, Phosphate Test)
NINHYDRIN TEST
- general test for protein (except proline: yellow); -NH2 group in free amino acid
Reagent: Ninhydrin Positive: deep blue/purple solution
Principle: Ninhydrin condense w/ammonia & hydrindantin to make Ruhemann’s purple

MOLISCHE TEST
- all carbo (except triose & tetrose)
Reagent: α-naphthol in ethanol, concentrated sulfuric acid
Positive: purple ring forms in between 2 layers.
Principle: H2SO4 to form furfural (aldehyde & organic compounds) & its derivatives
SALKOWSKI TEST
- cholesterol
Reagent: chloroform & concentrated sulfuric acid
Positive: depends on color (distinct & clear); bluish red to purple
Principle: cholesterol reacts w/sulfuric acid
TEST FOR PHOSPHATE
- phosphate; not specific for lecithin (phosphate w/lipids is positive to molybdate test)
Reagent: ammonium hydroxide, nitric acid, ammonium molybdate
Positive: yellow precipitates (phospholipid) Negative: fat & cholesterol
Principle: When phospholipids are added to strong acid solution, lipids hydrolyze,
producing free phosphate