Professional Documents
Culture Documents
Biochem Lab
Biochem Lab
BIURET TEST
- proteins
- Reagent: KOH/NaOH, hydrated CuSO4; Potassium sodium tartrate
- Positive: Violet solution
THEORETICAL RESULTS
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
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)
Glucosemaltosedextrinamylosestarch 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
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)
(+)
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
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