1. Identification of Some Macromolecules
1) What are 2 types of treatment controls, and explain each.
Positive control: gives positive result if experimental conditions were followed correctly
Negative control: gives negative result if experimental conditions were followed correctly
2) What are the most abundant elements in living material?
Carbon, Hydrogen, Oxygen, Nitrogen, Sulfur and Phosphorus
3) What are the 4 major biological macromolecules?
Carbohydrates (monosaccharides & polysaccharides), lipids, proteins, & nucleic acids.
4) What is the original colour of iodine, and what colour does it turn in presence of starch and glycogen?
Starch: yellow --> blue-black (amylase in starch reacts with iodine)
Glycogen: yellow --> reddish-brown (due to the multi-branched component)
5) How do plants and animals store simple sugar glucose?
In the form of polysaccharides. In plants, starch is the polysaccharide with glucose units linked by
glycosidic bonds. Glycogen, in animals, is a larger polymer.
6) How do starch and glycogen differ?
Molecular weight, overall shape and degree of branching in the final polysaccharide structure. Amylose
is unbranched and helical, where glucose units are joined by a(1-->4) linkages. Amylopectin has straight,
branched sections from a(1-->6) linkages. Glycogen is similar to amylopectin, but more highly branched.
7) What makes sugar a reducing sugar?
The terminal aldehyde group makes it an aldose sugar, which reacts in Benedict's test to make glucose a
reducing sugar. Blue solution will develop precipitate ranging from yellow, green, red or brown
8) What happens when benedict's solution mixes with solution with reducing sugar?
The blue cupric ions (Cu++) in benedict's solution get reduced to Cu+ by the terminal aldehyde group.
The amount of Cu O 2ormed is proportional to the concentration of free aldehyde groups.
4Cu+ + 2OH +2e --> 2Cu O 2 2H+ +2e
9) What gives a false positive reaction?
Presence of other substances that could be oxidized (i.e. ketose sugar --> fructose)
10) What test is done for proteins? And how does the colour change occur? Biuret test; The peptide bonds in the proteins with Cu++ ions and alkali give a violet peptide complex.
2. Isolation of Some Macromolecules
1) Why should the yeast be grinded with water and sand?
It will rupture the cell walls and cell membrane; the glucan (cellulose) in the cell walls, and glycogen,
proteins and NA in cytoplasm will be released.
2) What is TCA used for?
Polysaccharides (starch and glycogen) are soluble in TCA. Proteins and NA aren't, so they'll remain in
3) What is centrifugation?
A fractionation technique where centrifugal force sediments suspended particles at the bottom of the
Supernatant is liquid above the pellet.
The pellet is also precipitate.
Controlling the speed and time of a
centrifugal run will allow particles of
different sizes/properties to be
separated from the same suspension.
4) What is in the supernatant, and what is in the pellets?
Pellet: proteins and NA
5) What does NaCl do to the pellets?
Nucleic acids are soluble in strong NaCl, but protein isn't so it will remain in suspension.
6) What will boiling do to the protein? What will centrifugation do?
I will thicken (coagulate) the suspended protein molecules. Centrifugation will pack the proteins into a
pellet, and nucleic acids will be the supernatant.
7) Why is ethanol added to supernatant?
NA are insoluble in ethanol, so it will precipitate out of the solution to form a white suspension.
8) After centrifugation, H 2O 4s added to the pellets. Why?
NA are soluble, so it will go into the solution. 9) What will boiling the acid do?
It's a hydrolyzing process, so it will break up NA into component nucleotide subunits, and then
eventually into base and sugar and phosphoric acid subunits.
10) What's the difference between the boiled and unboiled NA solutions?
Boiled: Hydrolyzed NA
Unboiled: Unhydrolyzed NA
11) What will barium hydroxide do?
It carries out acid-base neutralization. A drop of each NA solution is placed on litmus paper. If
unchanged, it's still acidic. So, barium hydroxide is added drop by drop. Titration is reached when litmus
turns slightly blue--> slightly alkaline, and past a pH of 7. At the end of titration, barium sulfate will form
as white precipitate.
12) Why is pancreatic enzyme added to protein?
It will suspend the pellet, hydrolyzing the protein into amino acid subunits. In living cells, hydrolysis is
carried out by enzymes. Thymol is added to prevent bacterial growth.
13) What will phosphate buffer do to protein?
It will suspend the pellet, and it will be unhydrolyzed protein.
3. Characterization of Some Macromolecules
1) What is chromatography?
Technique that separates mixtures into their individual components. The solution is absorbed by the
cellulose fibers on the paper. The degree of absorption depends on the structure; the one that sticks the
most will be slowed down the most.
2) What is the stationary phase and mobile phase?
Stationary: the matrix (motionless substance such as cellulose)
Mobile: the solvent (influenced by solubility, molecular weight and polarity)
*If substance completely soluble --> moves fast, but no separation
*If insoluble --> no migration at all
3) When is chromatography terminated?
When the solvent has almost reached the opposite end of the matrix.
Rf = d travelled by substance/ d travelled by solvent (constant for any given substance in a particular
solvent system and matrix)
4) The proteins have gone through chromatography. What does ninhydrin-acetone do?
Reacts with amino acids to turn purplish-pink colour. 5) What do you see after chromatography of NA?
Under U.V. lamp, compounds with nitrogenous bases absorb the U.V. light to appear as a dark spot.
6) What categories do alanine, histidine, aspartic acid, lysine and methionine fall? Adenine, cytosine and
Those that have highest Rf values are hydrolyzed protein/NA; they're non-polar with hydrophobic
interactions (they have the greatest affinity to the mobile phase).
1) What is a spectrophotometer?
A white light source that is focused on a prism that separates white light into its distinct narrow portions
of the spectrum-bands of radiant energy. It measures the entire visible spectrum.
2) What is the relationship between the concentration light-absorbing solute and their absorbance?
They're proportional, according to Beer's Law (linear).
3) How do you create an absorption spectrum?
Read the absorbance of substance at different wavelengths to determine the maximum absorption.
Concentration curve is then made (% transmittance vs. concentration); Determine concentration of
4) Why are pigments deeply coloured?
Because it is unable to absorb wavelengths of that particular colour.
5) What are the major pigments?
Chlorophylls (a & b) and carotenoids (carotenes and xanthophylls).
6) Why are leaves of most plants green?
Chlorophyll molecules absorb all colours, except green; the green wavelengths are reflected back to us.
7) What regions in visible spectrum are most strongly absorbed by chlorophyll a & b?
a: 420 and 665nm
b: 455 and 640nm
8) How did the chromatogram look?
Top to bottom: carotenoids (orange), xanthophyll (yellow), chlorophyll a (bluish-green), chlorophyll b
(yellowish-green) 5. Enzymes
1) What are enzymes?
Biological catalysts and proteins.
2) What is their specificity determined by?
The number and order of their component amino acids and 3D configuration of protein.
3) Describe process of enzymes.
Substrate + Enzyme --> Substrate-enzyme complex --> product + enzyme
4) All enzyme-mediated reactions are reversible, BUT ....
direction of reaction depends on conditions, such as substrate & enzyme concentration, reaction time.
5) What is salivary amylase?
Digestive enzyme in saliva that acts on starch. It breaks off maltose from ends of starch chain and
consumes water. To digest substrate, it requires hydrolysis (hydrolytic reaction).
6) What is phosphorylase?
Enzyme that acts on starch by removing glucose molecules, rupturing glucose-glucose bonds, and
consuming phosphoric acid. (Phosphorylase -->Phosphorolysis). Phosphoric acid dissociates and energy
released creates glucose-phosphate bond in glucose-1-phosphate.
7) What is the direction of reaction mediated by phosphorylase determined by?
Relative concentrations of reactants and products.
8) What is starch synthase?
main enzyme that produces starch in an intact plant cell.
9) When will phosphorylase synthesize starch?
When enzyme attaches glucose molecule to starch primer molecule; makes it longer, giving a positive