Lab Exam Flashcards
Describe the general structure of proteins
High molecular weight polymers of many amino acids linked by peptide bonds to form a linear chain (called polypeptide). In solution they take on a 3 dimensional shape
_____ proteins exhibit only a secondary structure brought about by H-bonding within or between polypeptide chains
Fibrous proteins
Describe the structure of globular proteins
form 3-dimensional shapes as a result of interactions between R-groups of constituent amino acids. Exhibit tertiary level of structure
Proteins that are globular with more than one polypeptide chain are referred to as having ____ structure
Quaternary level of structure
Why would one need to know the concentration of a protein in solution?
To measure enzyme activity, to figure out nutritional state of tissue, etc
Colorimetric tests are based on what feature of copper ions?
Based on the principle that copper ions in alkaline solutions form purple-coloured complex with 2 or more peptide bonds, detectable by spectrophotometer.
How does the colorimetric test give an estimate of ONLY protein in a sample?
Free amino acids do not have peptide bonds for the copper ions to bind to and will therefore produce no colorimetric change
Why is albumin usually used to construct standard curves?
It is cheap, abundant, and available in purified form
The higher the concentration of protein in a colorimetric test, the _____ the colour
deeper
Of the two colorimetric tests used in lab one, the ____ is more sensitive
Lowry test
In lab one, how many standard curves were constructed?
- One for Biuret test, one for Lowry, and one for UV
The Biuret test was done at a wavelength of ____nm using ___ reagent
540nm, Biuret reagent
Why did we do dilutions of the unknown sample in Lab 1? What dilutions were used?
We did dilutions just in case the unknown sample was “off scale” with respect to the concentration curve in some way. Two fold dilution (1:1), ten fold dilution (1:9), and undiluted
The Lowry test was done at a wavelength of ____nm using ___ reagent
500nm using Lowry reagent and Folin reagent
Which three amino acids are detected using the UV test? Are any less easily detectable?
Tryptophan, tyrosine, phenylalanine (slightly less than the other two)
The UV test was done at a wavelength of ____nm using ___ reagent
280nm using NO reagents
What test from lab 1 would you use for a sample that you are trying to keep for a future experiment with a protein concentration around 0.05mg/mL?
IF the sample must be kept for the future, then no reagents can be use. The UV test is the best option
What test from lab 1 would you use for a sample that is known to be deficient in tyrosine?
The UV test would not be accurate, as it detects tyrosine, tryptophan, and phenylalanine. Biuret test or Lowry test would be better
What test from lab 1 would you use for a sample with a high protein concentration (approx. 10mg/mL) that there is a lot of?
Any test is possible, as long as appropriate dilutions are prepared
What test from lab 1 would you use for a sample that is turbid with an approx. protein concentration of 1 mg/mL?
Not UV, because turbidity affects UV results. High turbidity likely means the concentration is high so the Biuret test is the best candidate, though Lowry is possible after more dilutions
If you need a final volume of 3 mls of the unknown solution in order to take an absorbance
reading in a spectrophotometer, how would you prepare the unknown dilutions? In other words, how
much water and how much of the unknown solution would you need to pipette into a test tube to end
up with the proper volume (3 mls) and proper dilution (2-fold and 10-fold)?
Tube 1 - 3mL unknown
Tube 2 - 1.5mL unknown, 1.5mL water
Tube 3 - 0.3mL unknown, 2.7mL water
DNA in the nucleus is in a long linear _____, sometimes called a _______
Polynucleotide, called deoxyribonucleoprotein (DNP)
What is a “wheat germ”?
The vitamin-rich embryo of the wheat kernel (separated before milling)
Describe the three main steps for isolation of DNA
Homogenization - Tissue is ground up, blended, crushed to release molecule in question.
Centrifugation - Homogenate is spun to sediment debris, unbroken cells, nuclei. Leaves soluble molecules in the supernatant.
Enrichment of Target Molecule - Either pellet or supernatant must be treated in some way to make DNA (molecule of interest) more accessible.
How can one take advantage of DNAs molecular structure and general properties in order to isolate it? Describe the process
DNA is a large, linear molecule, insoluble at physiological pH and ionic strength, and it is tightly bound to protein. Because it is in the nucleus the cells and nuclei must be lysed, insoluble DNP sedimented by centrifugation, and then a salt solution added to cause DNA to dissolve. The protein precipitates and further centrifugation will sediment it. DNA in solution can be reprecipitated by adding ethanol and then carefully wound around a fork
What are some ways in which scientists can try to isolate molecules in cellular solutions?
Change ionic strength, add detergent, add organic solvents, separate using electric field, separate by filtration
Describe the function and components of the following solutions used in Lab 2: DNA buffer, TRITON-X, 6.5% NaCl, 95% Ethanol
DNA buffer - NaCl and sodium citrate (which prevents DNAse activity by chelating MG2+ ions)
TRITON-X - hydrophobic detergent that helps dissociate DNP and dissolve residual membrane debris
6.5% NaCl - Causes DNA to become soluble (dissolve) through agitation. Proteins will precipitate.
95% ethanol - Used to re-precipitate DNA into a solid mass
THE ____ method is arguable one of the most widely used methods to localize DNA in the nuclei of tissues for microscopic examination
Fuelgen method
Describe the reagent(s) used in the Fuelgen method of staining
A colourless version of basic fuchsin called leucofuchsin (referred to in this context as Schiff’s reagent)
Why were the liver tissue preparations in lab two submerged in 1N HCl
to hydrolyze only deoxyribose sugars of the DNA helix to allow Schiff’s reagent to bind to hydrolyzed sugars
What is the end result of the Fuelgen method?
The nuclei should stain purple/pink to make them easy to identify
What are different variables that affect stain intensity in the Fuelgen method?
Degree of hydrolysis (less hydrolysis, weaker stain - too much hydrolysis, some DNA dissolves)
Nucleolus will not stain (too much RNA)
_____chromatin will stain darker using the Fuelgen Method than ____chromatin
Heterochromatin, euchromatin
The Dische diphenylamine reaction takes advantage of DNA structure in what way? IS it a reliable test for measuring DNA?
Takes advantage of the fact that DNA molecules contain large amounts of deoxyribose sugars. the presence of partially hydrolyzed 2-deoxypentose sugars, diphenylamine reagent turns blue.
It is not completely specific to DNA but there is little interfering material present in cells. It is reliable
How does the UV spectrophotometric method of DNA quantification take advantage of the physical structure of DNA? IS this method reliable?
The flat ring structure of nitrogen bases absorbs light well in the UV wavelength of 260nm. This method does not require reagents but can be affected by turbidity. On top of this, DNA that is accidentally hydrolyzed will increase absorption. IF the sample is contaminated with other cellular components, they will ALSO increase absorbance (ex. proteins absorb 260nm light)
What is the “hyperchromatic effect” and why is it relevant for UV spectrophotometric quantification of DNA?
The phenomenon that hydrolyzed DNA increases absorbance at 260nm (moreso than non hydrolyzed DNA)
How does one determine if a DNA sample is “clean” of protein? IS this foolproof?
Measure UV absorbance at 260 and 280nm. 260 is ideal for DNA, and 280 ideal for protein, and the ratio of 260/280 in a pure DNA sample should be 2.05. if the ratio is greater than 1.85, the sample can be assumed to be clean. This is not 100% reliable because RNA also absorbs light at 260nm.
practice calculations lab 2/3
dnsaklds
You should note that the DNA present in your “crude” sample for lab 2/3 is only a small portion of the total mass of material extracted. What is the rest of the material?
As most of the membrane should have been dissolved by TRITON-X, the material is most likely composed of proteins and RNA
Why are mitochondria considered such an unusual organelle?
- Has two membranes serving very different functions (inner membrane has enzymes for respiration, outer membrane does not)
- Have a circular chromosome to direct protein synthesis
- Cellular respiration is clearly partitioned between matrix and inner membrane (citric acid cycle happens in matrix, ETC and ATP synthesis happen on membrane)
What enzyme is looked at in Lab 4?
Succinate dehydrogenase
The coenzyme of succinate dehydrogenase is ____ and they are both in what part of the cell?
FAD (flavin adenine dinucleotide), both tightly bound to inner mitochondrial membrane
Azide is a respiratory poison that blocks electron transport at the level of cytochrome oxidase in the respiratory chain. What effect would the omission of azide have on the rate of dye reduction in Lab 4’s experiment? Explain.
Lessen the rate of DCIP reduction as DCIP would no longer be the only electron acceptor. Without azide ETC would function normally so some of the electrons would be taken up by ubiquinone. This would resut in an inability to get accurate enzyme activity measurement. The addition of azide ensures that all of the electrons are taken up by DCIP.
Finish this equation:
Succinate + FAD —-(succinate dehydrogenase) —-> ___ and ____
fumarate, FADH2
_____ carries protons and electrons to the ETC, where _____ accepts them and passes them down the ETC to O2
FADH2,
ubiquinone
How does replacing ubiquinone in function allow for assay of succinate dehydrogenase activity? What else must occur?
Replaces ubiquinone with an artificial electron acceptor that does not complete the ETC. The ETC must be inhibited another way, which is why sodium azide is introduced (blocks transfer of electrons from cytochrome oxidase to O2), further funneling electrons to the artificial electron acceptor DCIP
How does the addition of malonate affect succinate dehydrogenase activity?
Malonate competes with the substrate succinate for the active site on succinate dehydrogenase. Should inhibit succinate dehydrogenase
The more succinate dehydrogenase activity there was in lab 4, the ____ the colour of the solution
less extreme
Why did we boil the pellet in one of the trials for lab 4?
To denature the enzyme succinate dehydrogenase
How did altering the concentrations of mitochondrial pellet change the results of the succinate dehydrogenase assay?
The more mitochondrial pellet there is, the more of the enzyme is present and therefore the more enzyme activity occurs
How did the addition of malonate affect the results of the succinate dehydrogenase assay?
Lowered rate of reaction
IN lab 4, higher absorbance equaled _____ enzyme activity
lower/decreased
The most important light absorbing pigment in plants is ____, which is found in the ____ of the cell
Chlorophyll, found embedded in the thylakoid membranes of chloroplasts of the cell.
What is the basic equation for photosynthesis?
6CO2 + 6H2O»_space;»light»» C6H12O6 + 6O2
The _____ is the primary step in photosynthesis. What occurs during this step and where in the cell does it occur?
Light reaction (aka Hill reaction). Photolyzes water, traps energy as ATP and NADP. Occrs in the thylakoid membrane of chloroplasts
What occurs during the “dark reactions” of photosynthesis? Where do they occur and what is another term used to describe them?
Products of the light reactions (NADP and ATP) are used to assist in the conversion of CO2 to glucose. Otherwise known as the Calvin cycle, they occur in the stroma of the chloroplast
What mechanism was used in lab 5 to measure rates of oxygen evolution?
oxygen electrode
How does an oxygen electrode function?
Cells are placed in a buffer solution in a chamber with a platinum electrode covered in teflon. A small current is passed through the elextrode and oxygen produced in the chamber diffuses across the membrane and is reduced at the electrode tip to form water. Higher concentration of oxygen = higher rate of diffusion across electrode membrane.
Why did we filter the algal suspension and take varying optical density measurements at the end of Lab 6?
To determine total pigment (chlorophyll a) concentration, which is necessary to calculate oxygen evolved/min/unit pigment
Why, specifically, were absorbance values recorded at 665nm, 650nm and 750nm at the end of Lab 6?
665nm and 650nm were for measurement of chlorophyll, but 750nm was as a correction for substances other than chlorophyll. Each reading at 665nm and 650nm were subtracted by the reading at 750nm before being used.
Given that some oxygen is consumed by algal respiration in experiment 5, how do you think this compromises any measurement of oxygen evolution?
Respiration would affect the rate of photosynthesis since a portion of oxygen produced during photosynthesis would be consumed during respiration. This would give an underestimate of photosynthesis referred to as “apparent photosynthesis”
Explain how you would expect temperature to affect the rate of oxygen evolution with reference to both the light and the dark reactions in photosynthesis during Lab 5
During dark reactions, high temperature should lead to high enzyme activity and therefore increased O2 consumption. However, when temperature gets too high, the enzymes used in the dark reactions should be denatured and therefore respiration rate should be very low. (light is not a limiting variable)
Given the fact that the herbicide you used in Lab 5 inhibits the light reactions of photosynthesis, explain the observed effect on oxygen evolution (be specific about the actual mechanisms in the light reactions that are being inhibited). If we were to use a herbicide that inhibited the Calvin cycle, how would this affect the rate of oxygen evolution?
The herbicide Terbutryn target phososytem II by binding to quinone. Over time this would affect O2 evolution by preventing the remainder of photosynthesis from occurring, not producing the necessary energy to initiate respiration.
Using an herbicide that inhibits the Calvin Cycle would inihibit Light reactions because NADPH and ATP would build up to such high sconcantrations that light reactions would be inactivated. This would result in no production of O2 and therefore an O2 evolution rate of 0
Under the bright light conditions used in this experiment, an increase in temperature usually results in an increase in the rate of oxygen evolution. If this experiment were performed under dim light conditions, would you expect the effect of temperature to be the same?
Under dim light the effect of temp on oxygen evolution would be lessened because there is less oxygen being produced. As the rate of ATP and NADPH by the light reactions would be the limiting factor not the dark reactions. There would still be an increase in O2 evolution and it would not be as great an increase as in bright light.
Eukaryotic cells are divided into functional compartments called ____
Organelles
What occurs during differential centrifugation?
Organelles and other subcellular components are separated based on relative mass and density by homogenization followed by centrifugation and various speeds to isolate the desire organelle
What was the purpose of Lab 6?
To isolate several subcellular fractions from yeast cells and determine which fraction contains the enzyme alkaline phosphatase
_______ are the membranous fraction of cells which includes the plasma membrane, endoplasmic reticulum, and Golgi complex.
microscomes
What is the function of enzyme alkaline phosphatase? What does it do in the context of this experiment?
Responsible for removing phosphate groups from many types of molecules, including nucleotides, proteins, and alkaloids. In this experiment it converts pnpp to pnp by cleaving a phosphate group. pnp absorbs light strongly at 410nm.
In lab 6, the _____ the absorbance of the solutions, the more enzyme activity is present
higher
At what wavelength were lab 6’s readings taken?
410nm
What are some reasons why Lab 6’s results may not be what was expected (ex. nuclear pellet + nuclear supernatant does not equal whole cell)
Alkaline phosphatase is not present in the nucleus or mitochondria, but instead is probably in another organelle present in the post-mitochondrial supernatant.
What was the purpose of vortexing the yeast cells in Lab 6 with the glass beads?
To lyse cells during vortex
The arrangement of the thylakoid electron transport chain promotes pumping of hydrogen ions from the _____ into the ______. What does this promote?
Stroma into the thylakoid space. Causes thylakoid lumen to become acidic. and stroma to become alkaline
What is the chemiosmotic hypothesis and how did we test it in lab 7?
The idea that the ETC in plants results in hydrogen ions accumulating in the lumen of the thylakoids in an ATP-generating process. Proton motive force drives protons through ATP synthase. In lab 7 we looked for pH changes that are a result of ATP synthesis in spinach chloroplasts
TRUE OR FALSE (if false, rewrite to make it true): In mitochondria, the transport of protons by the electron transport chain is from the intermembrane space into the matrix
False, the transport of protons by the ETC is from the matrix into the inter-membrane space
TRUE OR FALSE (if false, rewrite to make it true): Light promotes photosynthetic electron transport in the thylakoid membranes of chloroplasts, which causes protons to be actively translocated out of the lumen of the thylakoids into the chloroplast stroma.
False, it causes protons to be actively translocated out of the stroma into the lumen
TRUE OR FALSE (if false, rewrite to make it true): As the proton gradient increases, ADP synthesis is driven by the proton motive force thus generated.
False, as the proton gradient increases, ATP synthesis is driven by the proton motive force generated
Why is NaCl added to the pelleted chloroplasts in Lab 7?
To created a hypotonic solution, forcing the chloroplasts to swell and this allows pH changes in the stroma to be detected (membrane becomes leaky).
What must we do before performing the pH assay in lab 7? How did we do this?
Adjust concentration of chloroplasts so that it was 0.5 mg chlorophyll/mL. This is done by taking absorbance of 0.1mL chlorophyll with 19.9mL 80% acetone at 654nm. Absorbance was multiplied by 5.6 and diluted with NaCl until it reached desired concentration (C1V1 = C2V2)
IN lab 7, what is indicated by a pH increase?
H+ ions are being pumped from stroma into the thylakoid by ETC of light reactions ([H+] of medium reduced)
IN lab 7, what is indicated by a pH decrease?
Should occur when lights are off. pH of medium decreases because H+ move via ATP synthase into the stroma (and into medium via leaky membranes)
Explain how the addition of CCCP should affect Lab 7 results. How does it do this?
Allows H+ to run rapidly down the concentration gradient out of the thylakoid and into the stroma causing a decrease in the pH of the medium.
The ______ is the most widely accepted view for the structure of plasma membranes
Fluid mosaic model
What makes the fluid mosiac model unique in comparison to other models for plasma membranes?
IT states that proteins are not in separate layers but are in fact EMBEDDED in the membrane. Lipid bilayers are considered to be fluid, so phospholipids and proteins can exist in OR on them
Describe the permeability of the plasma membrane. How does this affect the rate at which molecules move through the membrane?
The plasma membrane is “semipermeable”, meaning it is most impermeable to large molecules, relatively impermeable to charged ions, and very permeable to lipid soluble, low molecular-weight molecules.
Molecules move through the membrane at differing rates. depending on their size and soubility
Movement of water across a plasma membrane is referred to as ____, whereas movement of solutes is referred to as ____
Osmosis, diffusion
Why is red onion used for the study of water movement in membranes?
IT has large, translucent cells surrounded by cell walls. Most of the volume of a cell is occupied by a central vacuole, which are purple/red in the onion’s outer layer and allow volume changes to be easily viewed under a microscope
What causes the red/purple pigment in red onions?
Anthocyanins in the vacuoles of the cells lining the outside of the onion
Distinguish between hypotonic and hypertonic solutions.
Hypotonic - Has solute concentration that is less than that inside the cell, causing water to rush into the cell (causes cell lysis in animal cells)
Hypertonic - HAs solute concentration that is more than that inside of a cell, causing water to rush out of the cell (causes plasmolysis in plant cells and shrivelling in animal cells)
Distinguish between plasmolysis and deplasmolysis
Plasmolysis - Process that occurs when plant cells lose water in a hypertonic solution (shrivelling of cytoplasm within cell walls)
Deplasmolysis - Swelling of the cytoplasm of a plasmolyzed cell
How does a plant cell in its natural, slightly turgid state react to being places in a solution high in “diffusable” solutes?
Initially, plasmolysis would occur, with water rushing out of the cell to maintain solute balance, but over time the solutes would enter the cell along the concentration gradient (either through diffusion or facilitated diffusion) and the cell would undergo deplasmolysis.
What is meant by the term “iso-osmotic threshold”
The point at which water balance is established in cells placed in a diffusable solute solution
In Lab 8, why was there a difference between the iso-osmotic threshold of NaCl and sucrose? What was the observed difference?
The difference was that the iso-osmotic threshold of the NaCl solution was 1/2 of that of the sucrose solution. This is because the NaCl solution is an ionic solution (enters the cell more readily), and the sucrose is non-ionic
If we were to use a solution of CaCl2 in lab 8, how would the isosmotic threshold of this solution compare to NaCl and sucrose? Explain your answer.
If CaCl2 was used, the iso-osmotic threshold would be 1/3rd of that of the sucrose and 2/3rds of that of the NaCl. This is because CaCl2 breaks down into 3 parts.
Explain how you could determine more precisely the isosmotic threshold for red onion vacuoles in lab 8?
By using 0.5M or 0.2M increments for each prepared solution
Polyethylene glycol (PEG) is a compound of very high molecular weight and does not enter the plasma membrane of the cells. You are asked by your boss to carry out an experiment in which pine cultured cells must experience water stress during a period of 50 days. On your lab bench you have a bottle of sucrose and a bottle of PEG. Which compound would you choose to add into the medium for your experiment? Defend your answer. (
If polyethylene glycol cannot enter the plasma membrane of cells, it would be the ideal choice for a water stress inducing medium, as the cells would not have a chance to regulate solute concentration gradient (by taking PEG in). This makes PEG more likely than sucrose to cause constant, unwavering water stress. Sucrose CAN enter the cell, and therefore a sucrose solution has a higher chance of allowing deplasmolysis
How are microtubules internally arranged and in what ways are they important?
Microtubules are composed of 13 protofilaments of alternating, staggered alpha and beta tubulin heterodimers. They are used in the cell’s cytoskeleton, and form cilia, flagella, and centrioles.
Describe the structure of flagella
Consist of 9 doublets of microtubules, 2 central microtubules, and several MAPs. Flagella are anchored to the cytoplasm via a basal body.
What was the purpose of lab 9?
To study polymerization of microtubules during regeneration of flagella in Chlamydomonas sp. Cells were deflagellated and flagellar growth was examined in comparison to assembly in the presence of colchicine and cycloheximide
What is the function of Colchicine in lab 9? What curve do we expect from it?
Colchicine binds to free tubulin dimers and prevents the addition of dimers to the microtubules. The effect is nearly immediate, but as colchicine is used up, flagella growth can occur again, producing a curve that starts off steady before rising.
What is the function of Cycloheximide in lab 9? What curve do we expect from it?
Cycloheximide inhibits protein synthesis, so while it takes some time to come into effect, it will continue to inhibit protein synthesis for a long time, and should produce a curve that rises slightly before levelling off.
What was the purpose of collecting flagellar-length measurements for the original culture (non-deflagellated cells) in lab 9?
To compare the increase in flagellar length for non-deflagellated cells to that of deflagellated ones. Initial flagellar length and ending flagellar length should differ between the two, indicating whether flagellar length is far below or above what would be “normal”
If there are 35 lines in one ocular micrometer unit using the 3.5x objective lens, how many mm and micrometers is that?
0.35mm, 350micrometers
If there are 3.5 lines in one ocular micrometer unit using the 40x objective lens, how many mm and micrometers is that?
0.035mm, 3.5micrometers
How was the algae deflagellated in Lab 9?
By lowering the pH of the algal medium to 4.0 by adding acetic acid, then restoring it to 6.8 using KOH
Before measuring flagellar length in the microscope in lab 9, you had to place a few drops of the culture into a tube containing Medium I and Lugol’s iodine. What was the purpose of this step?
Lugol’s iodine was added to kill and stain Chlamydomonas cells and flagella, preventing cells from swimming around (aroudn to find cells, VERY hard to measure flagella), and making them more visible than they would be without staining
How is a microscope calibrated?
Using a “stage micrometer” slide and the ocular micrometer of the microscope. The “0” lines of the ocular and stage microemeters must be lined up, and the number of stage micrometers lines it takes to go from 0 to 1 on the ocular micrometer are counted and recorded as “x lines = 1 ocular micrometer unit (omu)”
