BioChem Chp 5
What types of compounds make up the gels used in electrophoresis?
Agarose and polyacrylamide.
What are the two principal types of mass spectrometry?
Electrospray ionization (ESI-MS) and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF MS).
When would you choose to use a Potter-Elvehjem homogenizer instead of a blender?
If you needed to maintain the structural integrity of the subcellular organelles, a Potter-Elvejhem homogenizer would be better because it is more gentle. The tissue, such as liver must be soft.
If you had a protein X, which is a soluble enzyme found inside the peroxisome, and you wished to separate it from a similar protein Y, which is an enzyme found embedded in the mitochondrial membrane, what
Reasonably harsh homogenization would be able to liberate the soluble protein X from the peroxisomes, which are fragile. Centrifugation at 15,000 3 g would sediment the mitochondria (broken or intact). The supernatant would then have protein X but no protein Y. Freeze/thaw techniques and sonication would accomplish the same thing, or the mitochondria and the peroxisomes could be separated initially by sucrose-gradient centrifugation.
What is the basis for the separation of proteins by the following techniques? (a) gel-filtration chromatography (b) affinity chromatography (c) ion-exchange chromatography (d) reverse phase HPLC
(a) Size. (b) Specific ligand-binding ability. (c) Net charge. (d) Polarity.
What is an advantage of using an ELISA instead of a protein microarray to study a proteome? What is a disadvantage?
60. The advantages of an ELISA would be ease of use, low cost, and ready availability to any researcher. The disadvantages are that, compared to a microarray, relatively few proteins can be tested at one time. A microarray can test thousands of proteins in a single experiment, so it is much more powerful. However, it is also much more expensive and requires specialized equipment not readily available.
What is the purpose of the tag on the bait protein described in Figure 5.27?
64. The bait protein is constructed to have a particular affinity tag. The bait protein interacts with cell proteins of interest and then binds to an affinity column via the tag. In this way, the cell proteins of interest can be found and isolated.
What are two ways that a compound can be eluted from an affinity column? What could be the advantages or disadvantages of each?
A compound can be eluted by raising the salt concentration or by adding a mobile ligand that has a higher affinity for the bound protein than the stationary resin ligand does. Salt is cheaper but less specific. A specific ligand may be more specific, but it is likely to be expensive.
What are two ways that a compound can be eluted from an ion-exchange column? What could be the advantages or disadvantages of each?
A compound can be eluted by raising the salt concentration or by changing the pH. Salt is cheap, but it might not be as specific for a particular protein. Changing the pH may be more specific for a tight pI range, but extremes of pH may also denature the protein.
An amino acid mixture consisting of phenylalanine, glycine, and glutamic acid is to be separated by HPLC. The stationary phase is aqueous and the mobile phase is a solvent less polar than water. Which of these amino acids will move the fastest? Which one will move the slowest?
A nonpolar mobile solvent will move the nonpolar amino acids fastest, so phenylalanine will be the first to elute, followed by glycine and then glutamic acid.
What is the difference between a primary antibody and a secondary antibody?
A primary antibody is specific for a target protein that a researcher is looking for. A secondary antibody will react with the primary antibody. The secondary antibody carries the tag that makes the complex visible.
Gel-filtration chromatography is a useful method for removing salts, such as ammonium sulfate, from protein solutions. Describe how such a separation is accomplished.
A protein solution from an ammonium sulfate preparation is passed over a gel-filtration column where the proteins of interest will elute in the void volume. The salt, being very small, will move through the column slowly. In this way, the proteins will leave the salt behind and exit the column without it.
Sephadex G-75 has an exclusion limit of 80,000 molecular weight for globular proteins. If you tried to use this column material to separate alcohol dehydrogenase (MW 150,000) from b-amylase (MW 200,000), what would happen?
Both proteins would elute in the void volume together and would not be separated.
Assume that you are getting ready to do an amino acid sequencing experiment on a protein containing 100 amino acids, and amino acid analysis shows the following data:
Chymotrypsin would be a good choice. There are more than four residues of aromatic amino acids. The protein, containing 100 amino acids, would be cut four times, possibly yielding nice fragments roughly 20-30 amino acids long, which can be sequenced effectively by the Edman degradation.
Which enzymes or chemicals would you choose to use to cut the protein from Question 50? Why?
Cyanogen bromide would be useless, because there is no methionine. Trypsin would be little better, because the protein is 35% basic residues. Trypsin would shred the protein into more than 30 pieces, which would be very hard to analyze.
What types of macromolecules are usually separated on agarose electrophoresis gels?
DNA is the molecule most often separated on agarose electrophoresis, although proteins can also be separated.
What is the basis for the technique called ELISA?
ELISA is based on antibody-protein interactions. Specific antibodies, called primary antibodies, are put into microtiter plates to localize target proteins. A secondary antibody carrying some type of tag to render it visible is also added. If the target protein is there, then the combination of the protein- primary antibody-secondary antibody will be visible.
How could you isolate mitochondria from liver cells using differential centrifugation?
First homogenize the liver cells using a Potter-Elvejhem homogenizer. Then spin the homogenate at 500 3 g to sediment the unbroken cells and nuclei. Centrifuge the supernatant at 15,000 3 g and collect the pellet, which contains the mitochondria.
An amino acid mixture consisting of lysine, leucine, and glutamic acid is to be separated by ion-exchange chromatography, using a cation-exchange resin at pH 3.5, with the eluting buffer at the same pH. Which of these amino acids will be eluted from the column first? Will any other treatment be needed to elute one of these amino acids from the column?
Glutamic acid will be eluted first because the column pH is close to its pI. Leucine and lysine will be positively charged and will stick to the column. To elute leucine, raise the pH to around 6. To elute lysine, raise the pH to around 11.
Give an example of a scenario in which you could partially isolate a protein with differential centrifugation using only one spin.
If the protein were cytosolic, once the cells were broken open, you could centrifuge at 100,000 3 g, and all the organelles would be in the pellet. Your enzyme would be in the supernatant, along with all the other cytosolic ones.
Why is the order of separation based on size opposite for gel filtration and gel electrophoresis, even though they often use the same compound to form the matrix?
In a polyacrylamide gel used for gel-filtration chromatography, the larger proteins can travel around the beads, thereby having a shorter path to travel and therefore eluting faster. With electrophoresis, the proteins are forced to go through the matrix, so the larger ones travel more slowly because there is more friction.
What is the main difference between reverse phase HPLC and standard ion-exchange or gel filtration chromatography?
In most chromatography systems, the ligands and solvents are polar. In reverse phase HPLC, a solution of nonpolar compounds is put through a column that has a nonpolar liquid immobilized on an inert matrix. A more polar liquid serves as the mobile phase and is passed over the matrix. The solute molecules are eluted in proportion to their solubility in the more polar liquid.
What would happen during an amino acid sequencing experiment using the Edman degradation if you accidentally added twice as much Edman reagent (on a per-mole basis) as the peptide you were sequencing?
In the first cycle, the first and second amino acids from the N-terminal end would be reacted and released as PTH derivatives. You would get a double signal and not know which one was the true N-terminus.
How does HPLC differ from ion-exchange chromatography?
Ion exchange chromatography is a specific type of separation based on net charge of the molecules being separated. The term HPLC refers to chromatography procedures carried out under high pressure, but the basis of the separation could be ion exchange, gel filtration, reverse phase, or affinity chromatography.
Describe a procedure for isolating a protein that is strongly embedded in the mitochondrial membrane.
Isolate the mitochondria via differential or sucrose-gradient centrifugation. Use another homogenization technique, combined with a strong detergent, to release the enzyme from the membrane.
You are in the process of determining the amino acid sequence of a protein and must reconcile contradictory results. In one trial, you determine a sequence with glycine as the N-terminal amino acid and asparagine as the C-terminal amino acid. In another trial, your results indicate phenylalanine as the N-terminal amino acid and alanine as the C-terminal amino acid. How do you reconcile this apparent contradiction?
It is possible that your protein is not pure and needs additional purification steps to arrive at a single polypeptide. It is also possible that the protein has subunits, so multiple polypeptide chains could be yielding the contradictory results.
What useful information might you get if you did determine the N-terminal amino acid as a separate step?
It might tell you if the protein were pure or if there were subunits.
Amino acid compositions can be determined by heating a protein in 6 M HCl and running the hydrolysate through an ion-exchange column. If you were going to do an amino acid sequencing experiment, why would you want to get an amino acid composition first?
It would tell you a relative concentration of the various amino acids. This is important because it would help you plan your sequencing experiment better. For example, if you had a protein whose composition showed no aromatic amino acids, it would be a waste of time to use a chymotrypsin digestion.
With which amino acid sequences would chymotrypsin be an effective reagent for sequencing the protein from Question 50? Why?
It would work best if the basic residues were spread out in the protein. In that way, fragments in the proper size range would be generated. If all four of the basic residues were in the first 10 amino acids, there would be one long fragment that could not be sequenced.
What is the advantage of MALDITOF MS? 5-5 Protein Identification Techniques
MALDI-TOF MS is very sensitive and very accurate. Attomole (10218) quantities of a molecule can be detected.
A sample of a peptide of unknown sequence was treated with trypsin; another sample of the same peptide was treated with chymotrypsin. The sequences (N-terminal to C-terminal) of the smaller peptides produced by trypsin digestion were as follows: Met-Val-Ser-Thr-Lys Val-Ile-Trp-Thr-Leu-Met-Ile Leu-Phe-Asn-Glu-Ser-Arg The sequences of the smaller peptides produced by chymotrypsin digestion were as follows: Asn-Glu-Ser-Arg-Val-Ile-Trp Thr-Leu-Met-Ile Met-Val-Ser-Thr-Lys-Leu-Phe Deduce the sequence of the original peptide.
Met!Val!Ser!Thr!Lys!Leu!Phe!Asn!Glu!Ser! Arg!Val!Ile!Trp!Thr!Leu!Met!Ile.
Can you separate mitochondria from peroxisomes using only differential centrifugation?
No, peroxisomes and mitochondria have overlapping sedimentation characteristics. Other techniques, such as sucrose gradient centrifugation, would have to be used to separate the two organelles.
Of the two principal polymers used in column chromatography and electrophoresis, which one would be most immune to contamination by bacteria and other organisms?
Polyacrylamide.
What is the advantage to transferring proteins bands from a gel to nitrocellulose during the western blot?
Proteins are transferred to nitrocellulose because all of the protein ends up layered on top of a very thin membrane. This means that small volumes of the antibody solutions can be used to bind to the proteins. These antibodies are very expensive, so the less used, the better. Also, since the original proteins were imbedded in a gel, if the gel were reacted directly with the antibodies, the antibodies would not have easy access to the gel, as they would not percolate easily through the pores of the gel without the benefit of an electric current to push them.
What is proteomics?
Proteomics is the systematic analysis of an organism's complete complement of proteins, or its proteome. Just as we learned the basic dogma of molecular biology (DNA S RNA S protein), the technology now available has allowed scientists to describe all the DNA of an organism as its genome, all of the RNA as its transcriptome, and all of the proteins produced as its proteome. To understand the flux of proteins in a cell is to understand its metabolism.
Why do most people elute bound proteins from an ionexchange column by raising the salt concentration instead of changing the pH?
Raising the salt concentration is relatively safe. Most proteins will elute this way, and, if the protein is an enzyme, it will still be active. If necessary, the salt can be removed later via dialysis. Changing the pH enough to remove the charge can cause the proteins to become denatured. Many proteins are not soluble at the isoelectric points.
How does the addition of sodium dodecyl sulfate to proteins affect the basis of separation on electrophoresis?
SDS binds to the protein in a constant ratio of 1.4 g SDS per gram of protein. It coats the protein with negative charges and puts it into a random coil shape. Thus, charge and shape are eliminated.
What is meant by "salting out"? How does it work?
Salting out is a process whereby a highly ionic salt is used to reduce the solubility of a protein until is comes out of solution and can be centrifuged. The salt forms ion-dipole bonds with the water in solution, which leaves less water available to hydrate the protein. Nonpolar side chains begin to interact between protein molecules, and they become insoluble.
Design an experiment to purify protein X on an anion-exchange column. Protein X has an isoelectric point of 7.0.
Set up an anion-exchange column, such as Q-sepharose (quaternary amine). Run the column at pH 8.5, a pH at which the protein X has a net negative charge. Put a homogenate containing protein X on the column and wash with the starting buffer. Protein X will bind to the column. Then elute by running a salt gradient.
What physical parameters of a protein control its migration on electrophoresis?
Size, shape, and charge.
What does SDS-PAGE stand for? What is the benefit of doing SDS-PAGE?
Sodium dodecyl-sulfate polyacrylamide gel-electrophoresis. With SDS-PAGE, the charge and shape differences of proteins are eliminated so that the only parameter determining the migration is the size of the protein.
You are purifying a protein for the first time. You have solubilized it with homogenization in a blender followed by differential centrifugation. You wish to try ammonium sulfate precipitation as the next step. Knowing nothing beforehand about the amount of ammonium sulfate to add, design an experiment to find the proper concentration (% saturation) of ammonium sulfate to use.
Tables exist to tell you how many grams of ammonium sulfate [(NH4)2SO4] to add to get a certain percent saturation. A good plan would be to take the homogenate and add enough ammonium sulfate to yield a 20% saturated solution. Let the sample sit for 15 minutes on ice and then centrifuge. Separate the supernatant from the precipitate. Assay both for the protein you are working with. Add more ammonium sulfate to the supernatant to arrive at a 40% saturated solution and repeat the process. In this way, you will find out what the percent saturation in ammonium sulfate needs to be to precipitate the protein.
Why is it no longer considered necessary to determine the N-terminal amino acid of a protein as a separate step?
The Edman degradation will give the identity of the N-terminal amino acid in its first cycle, so doing a separate experiment is not necessary.
The accompanying figure is from an electrophoresis experiment using SDS-PAGE. The left lane has the following standards: bovine serum albumin (MW 66,000), ovalbumin (MW 45,000), glyceraldehyde 3-phosphate dehydrogenase (MW 36,000), carbonic anhydrase (MW 24,000), and trypsinogen (MW 20,000). The right lane is an unknown. Calculate the MW of the unknown.
The MW is 37,000 Da.
Why can the Edman degradation not be used effectively with very long peptides? Hint: Think about the stoichiometry of the peptides and the Edman reagent and the percent yield of the organic reactions involving them.
The amount of Edman reagent must exactly match the amount of N-termini in the first reaction. If there is too little Edman reagent, some of the N-termini will not react. If there is too much, some of the second amino acid will react. In either case, there will be a small amount of contaminating phenylthiohydantoin (PTH) derivatives. This error grows with the number of cycles run until the point that two amino acids are released in equal amounts, and you cannot tell which one was supposed to be the correct one.
What are two types of compounds that make up the resin for column chromatography?
The basis of most resins is agarose, cellulose, dextran, or polyacrylamide.
You wish to separate and purify enzyme A from contaminating enzymes B and C. Enzyme A is found in the matrix of the mitochondria. Enzyme B is embedded in the mitochondrial membrane, and enzyme C is found in the peroxisome. Enzymes A and B have molecular weights of 60,000 Da. Enzyme C has a molecular weight of 100,000 Da. Enzyme A has a pI of 6.5. Enzymes B and C have pI values of 7.5. Design an experiment to separate enzyme A from the other two enzymes.
The easiest way would be to use a sucrose gradient to separate the mitochondria from the peroxisomes first. Then break open the mitochondria via harsh homogenization or sonication, and then centrifuge the mitochondria. The pellet would contain protein B, while the supernatant would contain protein A. Contaminants could still exist, but they could be cleaned away by running gel filtration, on sephadex G-75 (which would separate enzyme C from enzymes A and B), and then by running ion-exchange chromatography on Qsepharose at pH 7.5. Enzyme B would be neutral and would elute, while enzyme A would stick to the column.
What are the main procedures involved in a western blot?
The first step in a western blot is the separation of proteins via electrophoresis. The next step takes the gel from the electrophoresis and transfers the proteins from the gel onto a thin membrane of nitrocellulose or other absorbing compound. Once the proteins are transferred, they are incubated with the primary antibody. Then they are incubated with the secondary antibody. Lastly, the bands are made visible by reacting with the substrates for the secondary antibody enzyme tag, or they are visualized with a fluorometer or X-ray paper.
What is the order of elution of proteins on a gel-filtration column? Why is this so?
The largest proteins elute first; the smallest elute last. Larger proteins are excluded from the interior of the gel bead so they have less available column space to travel. Essentially, they travel a shorter distance and elute first.
In reverse-phase HPLC, the stationary phase is nonpolar and the mobile phase is a polar solvent at neutral pH. Which of the three amino acids in Question 28 will move fastest on a reverse-phase HPLC column? Which one will move the slowest?
The nonpolar amino acids will stick the most to the stationary phase, so glutamic acid will move the fastest, followed by glycine and then phenylalanine.
What are some ways that the antibody-protein complex can be visualized in ELISA or western blots?
The protein-antibody complexes can be seen based on the nature of the tag carried by the secondary antibody. This tag may be an enzyme that produces a visible color when provided its substrates, a fluorescent marker, or a radioactive compound.
What differences between proteins are responsible for their differential solubility in ammonium sulfate?
Their amino acid content and arrangements make some proteins more soluble than others. A protein with more highly polar amino acids on the surface is more soluble than one with more hydrophobic ones on the surface.
What are some of the assumptions behind the logic of the experiment described in Figure 5.27?
There are many assumptions behind the experiment described in Figure 5.27. One must assume that the nature of the tag has not changed the binding of the protein. For example, if adding the tag makes a protein more likely or less likely to bind to it, then the conclusions about cellular protein binding may be incorrect. For example, one might conclude that two proteins bind together to serve their metabolic function, but this binding could be an artifact of the experimental conditions. One must also assume that tagging the proteins has not changed the affinity between the tag and the affinity column.
What is the point to labeling a secondary antibody with a marker that can be visualized instead of just labeling the primary antibody? 5-6 Proteomics
There are thousands of primary antibodies that can be purchased commercially. Other research requires a new primary antibody to be created. The process of attaching an enzyme, a fluorescent marker, or a radioactive compound is a long and difficult task. If every primary antibody had to be tagged, it would be a daunting exercise for the company trying to do it. Instead, a company can specialize in tagging a secondary antibody directed against goat, rabbit, or mouse antibodies. These secondary antibodies can then be used for any experiment where the primary antibody came from one of these animals. Also, there is a signal multiplication effect of using a primary antibody since several secondary antibodies will attach to a single molecule of primary antibody.
You are in the process of determining the amino acid sequence of a peptide. After trypsin digestion followed by the Edman degradation, you see the following peptide fragments: Leu-Gly-Arg Gly-Ser-Phe-Tyr-Asn-His Ser-Glu-Asp-Met-Cys-Lys Thr-Tyr-Glu-Val-Cys-Met-His What is abnormal concerning these results? What might have been the problem that caused it?
There are two fragments that have C-termini that are not lysine or arginine, which is what trypsin is specific for. Normally there would be only one fragment ending with an amino acid that was not Arg or Lys, and we would immediately know that it was the C-terminus. Histidine is a basic amino acid, although it is usually neutral and therefore does not react with trypsin. It is possible that, in the pH environment of the reaction, the histidine was positively charged and was recognized by trypsin.
If you had a mixture of proteins with different sizes, shapes, and charges and you separated them with electrophoresis, which proteins would move fastest toward the anode (positive electrode)?
Those with the highest charge/mass ratio would move the fastest. There are three variables to consider, and most electrophoreses are done in a way to eliminate two of the variables so that the separation is by size or by charge, but not by both.
Referring to Question 23, how would you purify protein X using ion-exchange chromatography if it turns out the protein is only stable at a pH between 6 and 6.5?
Use a cation-exchange column, such as CM-sepharose, and run it at pH 6. Protein X will have a positive charge and will stick to the column.
Apply A sample of an unknown peptide was divided into two aliquots. One aliquot was treated with trypsin; the other was treated with cyanogen bromide. Given the following sequences (N-terminal to C-terminal) of the resulting fragments, deduce the sequence of the original peptide. Trypsin treatment Asn-Thr-Trp-Met-Ile-Lys Gly-Tyr-Met-Gln-Phe Val-Leu-Gly-Met-Ser-Arg Cyanogen bromide treatment Gln-Phe Val-Leu-Gly-Met Ile-Lys-Gly-Tyr-Met Ser-Arg-Asn-hr-Trp-Met
Val!Leu!Gly!Met!Ser!Arg!Asn!Thr!Trp!Met! Ile!Lys!Gly!Tyr!Met!Gln!Phe.
Where did western blot get its name?
Western blot got its name as a humorous derivation from the original blotting technique called Southern blotting. The original blotting technique was for DNA and was developed by a researcher named Southern, so they called the technique the Southern blot. The next type of molecule to be blotted was RNA, so to distinguish from a Southern blot, it was called a northern blot. After that, the technique for blotting protein was developed and called a western blot.
What could be an advantage of using an anion exchange column based on a quaternary amine [i.e., resin-N1(CH2CH3)3] as opposed to a tertiary amine [resin-NH1 (CH2CH3)2]?
With a quaternary amine, the column resin always has a net positive charge, and you don't have to worry about the pH of your buffer altering the form of the column. With a tertiary amine, there is a dissociable hydrogen, and the resin may be positive or neutrally charged, depending on the buffer pH.
How can gel-filtration chromatography be used to arrive at an estimate of the molecular weight of a protein?
Within the fractionation range of a gel-filtration column, molecules elute with a linear relationship of log MW versus their elution volumes. A series of standards can be run to standardize the column, and then an unknown can be determined by measuring its elution volume and comparing it to a standard curve.
Referring to Question 19, could you separate b-amylase from bovine serum albumin (MW 66,000) using this column?
Yes, the b-amylase would come out in the void volume, but the bovine serum albumin would be included in the column bead and would elute more slowly.
What types of homogenization techniques are available for solubilizing a protein?
using a blender, a Potter-Elvejhem homogenizer, or a sonicator.
