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BIOL130L Study Guide - Cell Nucleus, Red Algae, Synapsis

Course Code
Heidi Engelhardt

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Summary for Quiz
Monday, May 30, 2005
12:13 AM
Identification of Some Macromolecules
Gist of Experiment
Use different tests to check for the existence of macromolecules in various substances
oIodine test checks for starch and/or glycogen
oBenedict's test checks for reducing sugars
oBiuret test checks for protein
Notes on Underlying Theory
The most abundant elements in living material are:
There are 4 major types of biological macromolecules:
Monosaccharides (i.e. glucose, fructose)
Disaccharides (i.e. sucrose)
Polysaccharides (i.e. starch, glycogen)
oNucleic acids
Iodine test
oInformation on starch:
It is a polysaccharide used by plants to store glucose
Glucose is held together with glycosidic bonds
It is a mixture of 2 different polymers: amylose and amylopectin
It is unbranched and helical molecule
The glucose is joined by alpha 1 -> 4 linkages
It is straight and highly branched
The glucose is joined by alpha 1 -> 6 linkages
oInformation on glycogen:
It is a polysaccharide used by animals to store glucose
Glucose is held together with glycosidic bonds
It is heavier than starch
It is similar to amylopectin in overall structure, but is more highly branched
oHow does the test work?
Iodine solution is usually pale yellow
It turns blue-black in the presence of starch because of the amylose
It turns red-brown in the presence of glycogen because of the multi-branched
Benedict's test
oInformation on sugar:

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All sugars can exist as straight chains or in ring form
The straight-chain forms are called aldose sugars
They have a terminal aldehyde group (C single-bonded to H, double-
bonded to O)
oHow does the test work?
Blue cupric ions (Cu++) in Benedict's solution are reduced to cuprous ions (Cu+)
by the free aldehyde groups, and we get a precipitate of cuprous oxide (Cu2O):
4Cu+ + 2OH- + 2e- -> 2Cu2O + 2H+ + 2e-
The amount of cuprous oxide formed is proportional to the concentration of free
aldehyde groups
The color of the precipitate varies depending on this as well (blue ->
green -> orange -> red -> brown)
Ketose sugars (i.e. non-aldose, which means non-straight chain, which means
no free aldehyde group) can ALSO reduce Benedict's solution
This happens because the basic conditions of the experiment isomerize
a ketose to an aldose, and then the reduction happens with an aldose
Biuret test
oInformation on proteins:
They are composed of amino acids, which are connected by peptide bonds
A peptide bond is the carboxyl group of one amino acid covalently linked to the
alpha-amino group of the next amino acid
oHow does the test work?
Biuret solution is a solution of sodium hydroxide (NaOH) and copper sulfate
Under alkaline conditions (caused by the NaOH), the peptide bonds within
proteins react with the Cu++ ions to form a purple complex
So we identify the presence of protein by looking for this purple color
Summary for Quiz
Monday, May 30, 2005
12:13 AM
Isolation of Some Macromolecules
Gist of Experiment
Use a variety of techniques to isolate macromolecules from a starting mixture
Experiment Procedure and Justification Thereof
1. We start with a yeast-sand mixture
a. Yeast cells have:
i. Glucan (a polysaccharide) in the cell walls
ii. Glycogen, proteins, and nucleic acids in the cytoplasm
b. Grind the yeast to rupture the cell walls and release all this stuff
1. Add TCA (trichloroacetic acid) and continue grinding
a. Polysaccharides (in this case glucan) are soluble in TCA, so they will go into solution
a. But the proteins and nucleic acids will stay suspended!
1. Centrifuge the suspension (so just the non-sand part)
a. When you do this on a liquid (remember the polysaccharides are suspended in the
liquid) with particles suspended in it (remember these are the nucleic acids and
proteins), all the suspended stuff goes to the bottom (it "sediments") and the liquid
remains on top
i. The sediment is known as the precipitate, also known as "pellet"
ii. And the top liquid stuff is the "supernatant"
1. Now we focus just on the pellet (i.e. the nucleic acids/proteins)
a. Add NaCl to the pellet
i. Nucleic acids are soluble in strong NaCl, so they go into solution!
ii. But the proteins remain in suspension

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1. Again we centrifuge this, and the proteins become the pellet, and the nucleic acids are the
1. Now we are going to SPLIT the nucleic acids and the proteins, and do stuff with each
Nucleic Acid Portion
1. Alright, first remember that we are dealing with a liquid here, because the nucleic acids were in
1. But the first thing we'll do is to add chilled ethanol, which will cause the acids to precipitate out of
solution to form a suspension
1. As before, we centrifuge to isolate the acids (which are in the pellet, of course)
1. But then we take the pellet and we add sulfuric acid, which makes the nucleic acids go into
solution again
1. Then we boil the stuff - but only ONE of the test tubes! (We have 2 test tubes' worth of nucleic
a. Boiling in acid is a "hydrolyzing process" - it breaks up the nucleic acid into the
nucleotide subunits, and then even FURTHER into the base and sugar and phosphoric
acid subunits!
1. So now we have one test tube of "hydrolyzed nucleic acid" and another of "unhydrolyzed nucleic
acid" Good times!
1. OK, remember we had sulfuric acid in there? Now we have to neutralize the solution! Details
a. We're going to use barium hydroxide, a base, to neutralize this solution
a. We're essentially going to perform a titration, where we use litmus paper to figure out
when the solution is acidic, when it is basic, and when it is neutral
a. The chemical formula is this: H2SO4 + Ba(OH)2 -> BaSO4 + 2H2O
i. Note that the precipitate (salt) which forms is barium sulfate - we will filter this
out later!
Protein Portion
1. OK, so remember that back in the day, we had protein and nucleic acid resulting from a
centrifugation…Well, now we're dealing with the protein portion, which is solid
1. We take half the protein and add pancreatic enzyme
a. This enyzme will hydrolyze the protein into its amino acid subunits
a. This simulates how the hydrolytic process is carried out naturally, because in real life it
is done with enyzmes!
1. And the other half of the protein we add phosphate buffer, which will not hydrolyze it at all!
1. To both we add thymol crystals, which prevent the growth of bacteria
Summary for Quiz
Monday, June 06, 2005
10:28 AM
Characterization of Some Macromolecules
Gist of Experiment
Use the method of chromatography to separate the proteins and nucleic acids earlier into their
individual components
Experiment Theory
Chromatography is a technique that separates mixtures into their individual components
oFor example:
If we put black washable ink onto a tissue, the ink will spread outwards from
the place where we blotted it
However, the various components of the ink can't all move at the same speed
as it spreads out - so the components will visibly separate
The pigment which moves the slowest will "stop" first, followed by the next
slowest, and so on…
The stationary phase in any chromatogram is the "matrix" - it is the substance onto which we
place the stuff to be measured (i.e. in the above example, it was the tissue - or more specifically,
the cellulose in the tissue which was reacting with the ink in the fibers)
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