Amino Acid Analysis/ Protein Analysis Flashcards
1
Q
What does amino acid analysis help determine?
What processes are involved?
A
Amino acid analysis helps to determine protein structure
▪ Analysis involves two processes:
- Separation of a mixture into components - Separation is based on the different properties of the side chains, such as polarity or charge. Separation is generally achieved by some form of chromatography.
- Detection of the components of interest - Detection is based on chemical reactions that generate coloured or fluorescent amino acid derivatives that can be seen and measured.
- can be qualitative (tells you what is present)
- can be quantitative (tells you how much is present)
- can be preparative (separated components can be recovered for further experiments)
2
Q
What is partition chromatography?
A
Partition chromatography is an important method for separating components of a mixture
- Particles of solid are chosen with a specific property, e.g. silica gel has HO-Si-OH groups that can hydrogen-bond to polar amino acids
- Stationary Phase
- Liquid solvent or buffer flows past the particles and is non-polar
- Mobile Phase
- Amino acids exchange (partition) between phases
- polar amino acids P spend more of their time hydrogen bonded to silica and move slowly
- non-polar amino acids N spend more time in solvent, and move almost as fast as solvent
- Silica gel is a polar molecule
- Select something nonpolar for the mobile phase
3
Q
What is thin layer chromatography?
A
- Silica gel is spread in a thin layer on a plastic sheet
- Samples are applied near the lower edge
- The lower edge is placed in solvent
- As solvent soaks up the sheet, different components of the sample move with the solvent at a different rate.
- The highest point reached by the solvent is the solvent front
- Each amino acid can be identified by its characteristic relative mobility RF
- Very polar amino acids have low RF, non-polar amino acids have high RF
- Different substances move at different rates, so the components of an initial mixture are separated. Pure samples of substances suspected to be in the mixture are also applied. Spots in the mixture can be identified if they move the same distance as one of the pure samples.
- Polarity is the basis for separation of substances by thin layer chromatography.
4
Q
In thin-layer chromatography what depends on the rate at which a sample will move up the sheet?
A
The rate at which a given sample, e.g. an amino acid, moves up the sheet depends on its relative preference for stationary phase (silica gel) or mobile phase (nonpolar solvent). A very polar amino acid such as aspartate will spend most of its time stuck to the silica gel and will barely move. A very non-polar amino acid such as leucine will spend most of its time in the solvent, and will move up the sheet almost as fast as the solvent. Amino acids with intermediate polarity will be in equilibrium between the two phases, and will move part way up the sheet.
5
Q
What is column chromatography?
A
- A granular solid such as silica gel is packed into a glass tube or column; silica is usually held in place by a porous disk at the bottom. A sample mixture is applied at the top, and then solvent or buffer solution is allowed to flow through. Sample solutes travel with the flow of buffer solution to the bottom of the column. Substances that bind more strongly to the solid phase require more buffer to pass through or be eluted from the column. The main advantage is that the separated components of the mixture can be collected allowing additional experiments to be performed on the individual components
- Volume of buffer needed to move a compound through the column is the elution volume. Compounds can be identified by their characteristic elution volume
6
Q
What is high-performance liquid chromatography (HPLC)?
A
Column chromatography using specially designed columns and with solvent pumped through for greater efficiency. This is the usual method in research labs.
7
Q
How are amino acids detected?
A
- Amino acids are colourless, and samples may be 10-6 to 10-10 moles
- They can be detected by adding ninhydrin which reacts with primary and secondary amines
- Gives intense purple colour (10-8 moles detectable), or yellow colour for proline
- Spray ninhydrin onto TLC plates, or add to amino acid solution, and heat
- Colour intensity is proportional to quantity of amino acid, and can be measured
- Alternative is fluorescamine, giving yellow fluorescence under UV light (10-10 moles detectable)
- An alternative method often used in conjunction with HPLC is to prelabel the sample compound with a coloured or fluorescent dye before separation, and record the color intensity as each amino acid emerges from the column. This method is often preferred for quantitative analysis, since the dye reaction can be allowed to go to completion ahead of time.
- Dyes used for prelabelling include fluorodinitrobenzene, dansyl chloride, dabsyl chloride, phenylisothiocyanate
- Ninhydrin and fluorescamine can’t be used to label amino acids before separation since the color- forming reaction destroys the amino acid.
8
Q
What is Reverse Phase chromatography?
A
Instead of polar silica gel, a non-polar hydrocarbon silicon derivative is used as the solid stationary phase; instead of non-polar solvent, polar solvent is used as mobile phase. The order of passage is reversed, since now polar solutes don’t bind and have high RF, while non-polar solutes do bind and have low RF. (Used because it’s better at distinguishing subtle differences in hydrocarbon side chains of amino acids.)
9
Q
What is Ion Exchange chromatography?
A
- Ion exchange chromatography separates on the basis of charge
- Uses charged resins as stationary phase
- Cation exchanger resins contain negative groups, which bind positive molecules (cations ) - Cation Exchange Chromatography
- Anion exchanger resins contain positive groups, which bind negative molecules (anions) – Anion Exchange Chromatography
- Elution is by:
- Competition with a high ion concentration (usually NaCl), which displaces the amino acid from the resin
- Changing the pH to alter the charge on the amino acid, so it no longer binds to the resin
The silica gel or cellulose stationary phase is replaced by ionic resins
Solutes will now bind according to their charge rather than polarity, e.g. positive amino acids NH3+-CHR-CO2H bind to negative charged resin.
- At pH 2.5, ⍺-amino groups exist as NH3+ while ⍺-carboxylate groups exist as 50% COO- & 50% COOH giving the amino acid an overall positive charge
- Side chains can also contribute to the charge
- The exact value of overall charge depends on specific pKa values of the various groups in each amino acid
- Size of net charge determines how tightly each amino acid binds
- High Na+ present in elution buffer first displaces weakly bound amino acids. As [Na+] is increased, more tightly bound amino acids are progressively displaced
- Alternatively, pH may be increased to eliminate the positive charge on the amino acid, so it no longer binds to the resin
10
Q
how can amino acids be separated by ion exchange?
A
- Amino acids are detected and their concentration measured in buffer coming out of the column
- Elution volumes are often compared relative to a common standard, such as Ala or Leu
- Elution volumes are characteristic for each amino acid, and allow them to be identified
- The volume of buffer needed to move a given amino acid from the top to the bottom of the column is the elution volume
11
Q
Explain the separation of proteins from complex mixtures?
A
- Proteins are derived from natural sources such as microbial cultures, plants, or animal tissues such as liver
- Extracts may contain thousands of different proteins
- Separation by ion exchange is based on charge differences among proteins
- depends on the relative number of Asp + Glu (negative) versus His + Lys + Arg (positive) in each protein, and on pH
- ~65% of all proteins are negatively charged at pH 7
- Complete protein purification involves successive application of several chromatographic or other separation techniques. Since there may be other proteins with similar charge, separations based on other properties such as size are also applied.
12
Q
What are the charge differences among peptides and proteins?
A
- Peptides and proteins can show large differences in charge
- Ion exchange is frequently used to separate protein mixtures
- Anion exchangers are positive charged polymers that bind and retain negative charged solutes (anions) including proteins.
- Cation exchangers are negative charged polymers that bind positive charged solutes(cations) including proteins.
13
Q
What does the upper and lower part of this graph show?
A
Proteins with the appropriate charge will bind to ion exchanger. They are released from the resin by gradually increasing the concentration of neutral salt such as NaCl or KCl, a technique known as gradient elution.
- The upper part of the graph on the right shows a gradually increasing NaCl concentration in the buffer.
- The lower part shows the protein concentration measured in the buffer as it comes out of the column.
14
Q
What determines how quickly a protein will elude?
A
- Proteins that lack charge or have the same charge as the resin will not bind and are eluted quickly. Proteins that have the opposite charge to the resin bind until the NaCl concentration has risen enough to release them.
- Proteins that have a higher charge will bind more tightly, and a higher NaCl concentration is needed to release or elute them. Resin and pH are chosen so that the desired protein binds moderately tightly.
15
Q
What is metal affinity chromatography?
A
- Clusters of His in a protein bind tightly to Ni2+ or Co2+
- Column is made up of chelating resin containing Ni2+
- If protein is artificially produced by inserting its gene into cells, the gene can be modified to include 6-8 extra His residues at N- or C-terminus
- The added His cluster is called a His-tag
- His-tagged proteins binds tightly to the Ni2+ resin
- The His-tagged protein is eluted by adding imidazole (structure similar to His side chain) to the buffer
- Imidazole will occupy the Ni2+ sites on the resin, allowing the His-tagged protein to pass out of the column and be collected.
- High degree of purification in one step
Natural protein: Met-Pro-Ser-Leu-Ser-Tyr-etc
His-tagged protein: -His-His-His-His-His-His-Pro-Ser-Leu-Ser-Tyr-etc
17
Q
What is polymeric gel?
What size proteins can enter? Which can not?
A
Beads of polymeric gel - a loose network of polymer with many water-filled pores.
- Protein molecules can enter the pores if they fit
- Larger proteins are excluded from the pores
- Proteins of intermediate size may enter some of the pores
- The stationary phase of the column consists of a hydrated gel, formed from a polymer, which absorbs water to form an aqueous network of open pores.
- The gel is in the form of beads or granules. Molecules in the sample that are small enough can fit into pores the gel. Since the gel is the stationary phase, these molecules progress through the column more slowly.
18
Q
What is column chromatography using gel filtration?
A
- Small molecules enter pores and are delayed in their movement down the column
- Intermediate size molecules can only enter some pores and are delayed less
- Very large molecules are excluded from gel and stay in the buffer flowing around the beads – pass through the column quickly
19
Q
How can gel filtration be used to measure the molar mass of proteins?
A
- Measure elution volume of proteins of known mass
- Elution volume Ve is the volume of buffer needed to move a protein from top to bottom of column
- Elution volume is a linear function of log molar mass (negative slope)
- Then measure elution volume of unknown protein and project back to the log mass axis
- If the elution volume is measured for two or more proteins of known molecular mass, it is then possible to measure the elution volume of an unknown protein and estimate its molecular mass either by interpolating the graph or by deriving the equation of the straight line.
21
Q
How can you separate proteins using electrophoresis?
A
- Electrophoresis is separation based on movement of charged particles in an electric field.
- A mixture of proteins is placed between a pair of electrodes immersed in a conductive buffer solution, and a voltage of 100-1000 V applied.
- Positive molecules move towards the negative electrode and negative molecules move to the positive electrode.
- The rate of movement is a function of size, shape, and charge
- To prevent movement of the solution, the separation is carried out in a porous gel
22
Q
What is Polyacrylamide gel?
A
- Polyacrylamide gels are easily formed from simple chemicals in the lab, and are often used for proteins where molecular mass is in the range 10-1000 kDa.
- Since free solution is subject to disturbances by convection (local fluid motion caused by temperature differences), the buffer is immobilized in a gel.
- On the molecular scale, the gel is sufficiently porous to allow protein sized-molecules to pass through. Agarose gels are best for very high mass, especially DNA where molecular masses may be >10 MDa.
- A typical gel is 5-15% polymer and 90-95% water with conductive buffer
23
Q
Explain SDS-Polyacrlamide Gel Electrophoresis (SDS-PAGE)
A
- a modified form of electrophoresis in which protein is treated with the ionic detergent sodium dodecyl sulfate, SDS.
- SDS ions coat the protein molecules, which adopt rodlike shapes, so that with SDS bound, all proteins have the same rodlike shape
- The strong negative charge of the bound SDS ions overrides the somewhat variable charge of the polypeptide itself
- small proteins can fit through all the pores, move rapidly down the gel
- large proteins have to “meander” to find pores to fit through, move more slowly
- The charge of the complex then depends on the number of SDS molecules bound, which in turn depends on the size of the polypeptide. As a result, all polypeptides now behave as if they had similar charge per unit length.
- SDS-PAGE may also be used to measure the molecular mass of a polypeptide chain by comparing with standards of known size
24
Q
Isoelectric focussing: How do you separate based on Isoelectric Point of proteins
A
- Every protein has an isoelectric point, the specific pH at which the sum of negative charges is exactly equal to the sum of positive charges and its net charge is zero.
- At high pH, protein is deprotonated, moves toward the + electrode
- If the positive electrode is placed at the low pH end of the gradient, the protein migrates towards the positive electrode, and passes through buffer of gradually decreasing pH.
- As the pH decreases, different side chains in the protein become protonated, causing the net negative charge to decrease. At some point the protein reaches the pH equal to its isoelectric point, where it has no charge and stops migrating since there is no attraction to either electrode. Separation occurs because each protein in a mixture has a different isoelectric point.
24
Q
What is Two-dimensional electrophoresis?
A
- Involves separation of a protein first by isoelectric focussing in a thin capillary tube. The spaghetti-like gel contains the partly separated proteins, and is then laid on the top edge of a conventional SDS-PAGE gel. A second separation by electrophoresis is then carried out at 90 ̊ to the original isoelectric focussing.
- Combines isoelectric focusing and SDS electrophoresis
- Can separate individual proteins in very complex mixtures
25
Q
What is Mass Spectrometry?
A
- Is a technique often used in conjunction with electrophoresis to identify proteins
- A protein is vaporized by laser beam, yielding charged protein particles
- Particles travel toward the detector
- Velocity depends inversely on the mass of the particle (larger = slower)
- The time of flight to the detector yields a very accurate mass measurement (5 significant figures of accuracy)
- We can compare the mass of the protein with a database of proteins of known mass, allows identification
28
Q
What is a nucleophile?
A
- A nucleophile is simply an atom with a lone pair of electrons that is available to share with another nucleus.
- Nucleophiles seek out other groups that are electron-deficient (“nuclei”)
29
Q
How may an atom with a lone pair use it?
A
- As an H-bond acceptor if it simply attracts an O-H or N-H
- R-O: - - - H–N-R
- As a base if it captures H+
- H+ + :NH2–R → +NH3–R
- As a nucleophile when it shares the lone pair with another electron-deficient atom to make a new bond
- A nucleophilic displacement is a reaction in which an incoming nucleophile X: attacks a target atom C to displace another attached group. The group Y that detaches is called the leaving group
- X: C––Y → X––C :Y
Nucleophilic Addition: X: C=Y → X–C–Y
30
Q
Hydrolysis is attack by H2O as nucleophile on the electron deficient C of the peptide bond (Nucleophilic Displacement)
A
- C is electron-deficient (an electrophile; electron-lover), because the electronegative O bonded to C pulls electrons away from it
- this lets C take up the incoming electron pair in a new bond
- Curly arrows represent movement of electron pairs
- The transition state is semi-stable, like a compressed spring
- Amino N acts as leaving group, allowing the peptide bond to break
32
Q
How is the linear chain of myoglobin folded?
A
- The linear chain of myoglobin is precisely folded into a compact, 3-dimensional globular structure
- A simplified view shows distinct regular patterns in folded protein
- The ribbon traces the path of the polypeptide backbone (peptide bonds only, no side chains)
- Colour coding: blue at N-terminal progressing to red at C-terminal
- In myoglobin, the ribbon is arranged in eight spiral or helical segments
- Amino acid side chains fill the “spaces”, which are not empty
33
Q
What are the three (or four) organization levels of protein structure?
A
- Primary structure: the linear sequence of amino acids
- Secondary structure: regular repetitive patterns, such as the helical sections in myoglobin, that run along short sections of peptide chain (5-20 AAs)
- Tertiary structure: the overall pattern of 3D folding of the whole polypeptide
- in myoglobin, 8 helical sections enclose a central cavity
- Some proteins also have a quaternary level of structure
- e.g. hemoglobin is an assembly, or complex, of four globin units, each one similar to myoglobin
- the four globins act cooperatively to improve oxygen transport
- Protein function depends on the positioning of key amino acids close to each other in 3-D space, even though they are far apart in the linear sequence.
- The exact pattern of folding is critical for protein function.
