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.
