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BIOCHEMISTRY > Protein Analysis > Flashcards

Protein Analysis Flashcards

1
Q

It is a separation technique that exploits various properties of biomolecules.

A

Chromatography

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2
Q

Chromatographic separation may be based on the molecules’:

A 
Size
Shape
Hydrophobicity / hydrophilicity
Charge
Affinity to a ligand
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3
Q

A form of adsorption chromatography in which charged molecules are exchanged for small ions of like charges.

A

Ion-Exchange Chromatography

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4
Q

These are chemical structures where ions are attached.

Attached to a variety of solid supports such as cellulose, agarose, and vinylbenzene

A

Ion exchangers

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5
Q

Ion exchangers Examples:

A

Anion exchangers

Cation exchangers

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6
Q

It exchange negatively charges ions.

A

Anion exchangers

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7
Q

It exchange positively charged ions or cations.

A

Cation exchangers

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8
Q

Ion-Exchange Chromatography

The amino acids are the following:

A

Glutamic acid
Glycine
Lysine

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9
Q

pI Value:
Glutamic acid
Glycine
Lysine

A

-

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10
Q

It is the content of the column used in ion-exchange chromatography where the sample was applied.

A

3.4 pH citrate buffer

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11
Q

The first 12 tubes were then labeled and filled with __ coming column.

A

3ml of the eluent

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12
Q

Which of the amino acids will you expect to come out of the column? Explain your answer

A

Glutamic acid will be the first amino acid to come out of the column. At this stage of the experiment, we are using the 3.4 pH citrate as our buffer. At this pH, the glutamic acid becomes deprotonated at its α–COOH group, loosing its net positive charge. As a consequence, the amino acid will not be adsorbed by the cation exchanger and will instead be washed by the eluate.(Recall that the isoelectric pH of glutamic acid is 3.22)

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13
Q

Another set of 12 tubes were prepared, some contents of first set (A) was transferred to this second (B) set.

A

The contents of the B set of tubes are to be tested.

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14
Q

Which of the following test is suitable for detecting amino acids? Explain the principle of the test.

A. Biuret test
B. Bromcresol purple dye binding test
C. Ninhydrin test

A

Ninhydrin test is the best suitable method for detecting amino acids. The first two techniques are only used to detect peptide bonds (or specific proteins).

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15
Q

This test is a general test and thus given by all amino acids.

This test is due to a reaction between a amino group of free amino acid and ninhydrin.

A

Ninhydrin test

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16
Q

It is a powerful oxidizing agent and its presence, amino acid undergo oxidative deamination liberating ammonia, CO2, a corresponding aldehyde and reduced form of ninhydrin (hydrindantin).

A

Ninhydrin

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17
Q

It is a blue substance formed when the NH3 formed from a amino group reacts with another molecule of ninhydrin and is reduced product (hydrindatin).

A

Diketohydrin (Ruhemanns complex

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18
Q

Ninhydrin Reaction

A

SEE anki

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19
Q

It is a test that allows chemists to chemically detect amino acids.

A

Ninhydrin Test

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20
Q

What are the possible clinical / forensic applications of this technique?

A
  1. Used by forensic personnel to visualize fingerprints on surfaces. This is possible due to the minute amounts of amino acids that collect in an individual’s unique fingerprint ridges.
  2. Detection and quantification of amino acids in clinical specimens such as cerebrospinal fluid, serum, urine after separation.
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21
Q

The tubes with the heaviest reactions were noted and their corresponding set A tubes are saved.

A

Now with the first 12 tubes are done, it is now time to shift to the 2nd buffer.

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22
Q

For better resolution of the procedure, which of the following buffers should be used? Explain your answer.

A. Citrate buffer at pH 6.2
B. Sodium carbonate buffer at pH 11

A

Citrate buffer at pH 6.2 is the 2nd buffer to be used in this experiment. In doing ion-exchange chromatography, it is important that we gradually adjust pH for better separation of amino acids.

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23
Q

What will happen if you abruptly change the pH used for the second buffer?

A

Abrupt change in pH (in this case, immediately switching to the most basic buffer) will result in all the remaining amino acids to deprotonate and loose their positive charges, causing them to elute, resulting in a poor resolution of the technique.

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24
Q

The buffer was now switched to citrate at 6.2 pH and was subsequently allowed to run down the column.

A

Ok

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25
Q

After buffer application which of the following amino acid will elute (washed off) from the column?

A. Glycine
B. Lysine

A

A. Glycine

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26
Q

Why glycine will be eluted from the column after using 6.2 pH citrate buffer?

A

Glycine will elute from the chromatography column after changing the pH to 6.2. Glycine has an isoelectric pH of 5.97, changing the pH from 3.4 to 6.2 will render the amino acid electrically neutral and therefore elute from the cation exhanger.

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27
Q

The second 12 tubes (13 – 24) were then labeled and filled with 3ml of the eluent from the column (set A).
Another set of 12 tubes was prepared (set B).
A sample of the contents of tubes 13 to 24 where transferred to the corresponding set B tubes.
The set B tubes were tested for the presence of amino acids with ninhydrin.

A

Ok

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28
Q

The tubes with the heaviest reactions were noted and their corresponding set A tubes are saved.
Now with the second 12 tubes are done, it is now time to shift to the 3rd buffer.
The buffer was now switched to sodium carbonate at pH and was subsequently allowed to run down the column.

A

Ok

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29
Q

The last remaining amino acid in the column is lysine

A

Ok

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30
Q

Why does lysine require a more basic buffer for it to elute?

A

Lysine has an isolectric pH of 9.79. At lower pH, the amino acid will have a net positive charge and will remain adsorbed on the resin particles containing the cation exchanger. Also, changing the pH to 11 had the effect of deprotonating all the amino and carboxylic groups in the lysine molecule, giving it a more negative charge which will be repelled by the cation exchanger.

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31
Q

The last 11 tubes (25 – 35) were then labeled and filled with 3ml of the eluent from the column (set A).
Another set of 11 tubes was prepared (set B).
A sample of the contents of tubes 25 to 35 where transferred to the corresponding set B tubes.
The set B tubes were tested for the presence of amino acids with ninhydrin.

A

Ok

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32
Q 
The tubes with the heaviest reactions were noted and their corresponding set A tubes are saved.
Three tubes (one each amino acids) will be saved and used subsequently for the next experiment.
A

Ok

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33
Q

What are the applications of ion-exchange chromatography in the clinical or preclinical research settings?

A
  1. Analysis of inorganic ions.
  2. Analysis of nitric oxide (chemical messanger).
  3. Analysis of heavy metals in clinical specimen.
  4. Detection of mono- and oligosaccharides, alditols, amino acids and peptides.
  5. Pharmaceutical research.
  6. Analysis of purity, content and stability of pharmaceutical products.
  7. Used as a separation technique for various biochemical studies.
34
Q

It uses molecular size to fractionate biomolecules.

A

Gel filtration

35
Q

Gel filtration

Stationary phase:

A

Gel consisting of beads containing pores of a defined and narrow size range

36
Q

What will happen to the biomolecules that are applied in the gel?

A

These will allow only biomolecules below a particular mass to diffuse into the pore. As the sample progresses through a bed of such stationary phase, smaller molecules will be retained more readily than larger ones. Molecules of greater mass than the exclusion limit will pass freely through the column, eluting in a single, unresolved, peak at the void volume. Molecules within the fractionation range of the gel, however, will be retained in a manner generally inversely proportional to their mass, that is smaller molecules will be retained longest.

37
Q

The fractionation range and resolution achievable with such a chromatography system will depend greatly on the?

A

Composition and uniformity of the population of beads in the stationary phase.

38
Q

Applications of gel filtration in biochemistry:

A
  1. Removal of low-molecular weight components in samples (desalting).
  2. Determination of protein molecular masses.
  3. Protein purification.
39
Q

It separates soluble proteins by using reagents that disrupt solvation, exposing their hydrophobic groups which interacts with a hydrophobic stationary phase.

A

Hydrophobic interaction

40
Q

It utilizes a protein’s ability to a ligand as the basis for separation (example: isolating an enzyme or an antibody from a sample using a chromatography column with embedded ligand, which can be a substrate or an antigen). Because of the highly specific nature of biological recognition phenomena, only the molecule to be purified will bind to the stationary phase and other molecules will pass freely through the chromatography system.

A

Affinity

41
Q

The metals are immobilized on a stationary phase and metal-binding regions of proteins bind to this ligand.

A

Immobilized metal affinity chromatography

42
Q

Amino acid side chains most likely to coordinate to metals are those of?

A

Histidine, cysteine and tryptophan.

43
Q

It uses alkyl chains covalently attached to the stationary phase particles. Employs polar solvents such as water, methanol, acetonitrile or tetrahydrofuran or mixtures of these.

A

Reversed phase

44
Q

In reversed phase chromatography, __ will be retained by the stationary phase, which can be eluted by __.

A

Hydrophobic molecules

Nonpolar solvents

45
Q

Reversed phase chromatography separates molecules based on their?

A

Hydrophobicity

46
Q

It operates at atmospheric pressure, using stationary phase particles with relatively large (e.g. 50–150 µm) diameters. Gives relatively low resolution and is slow compared to the more high-resolution systems described in the following sections.

A

Open column chromatography

47
Q

Advantage of Open column chromatography

A

Stationary phases and other equipment used are cheap .

48
Q

It uses smaller sized column particles in combination with higher pressures to achieve higher resolution.

A

High performance liquid chromatography (HPLC)

49
Q

By reducing the transit time on the column, HPLC can limit __and thus greatly improve resolution.

A

Diffusional spreading of sample bands

50
Q

What is the use of HPLC?

A

Separation and analysis of low molecular weight biomolecules (metabolic intermediates, building blocks of biopolymers, and analysis of peptide mixtures)

51
Q

It similar to HPLC but operates at lower pressures.

A

Fast protein liquid chromatography

52
Q

In fast protein liquid chromatography, what is the solvent and stationary phase used?

A

Aqueous solvents

Stationary phases have a relatively large loading capacity

53
Q

What is the use of fast protein liquid chromatography?

A

It used to isolate not only proteins but biomolecules including DNA (plasmids, restriction fragments and oligonucleotides), nucleotide derivatives (e.g. NAD, ATP), carbohydrates as well as peptides and other low molecular weight biomolecules.

54
Q

It involves a stationary phase particle which permits the use of very high flow rates with little back-pressure.

A

Perfusion chromatography

55
Q

The stationary phase particle in perfusion chromatography contain?

A

Large channels (1μm) which facilitates rapid diffusion of the mobile phase, thus limiting mass transfer effects that decreases the resolution of the technique at high flow rates.

56
Q

The stationary phase is composed of thin, porous membranes which are layered on top of each other. These may be coated with ion exchange, affinity or other groups, providing a range of chromatography modes.

A

Membrane-based chromatography

57
Q

The technique involves a gaseous analyte which is transported through the column by a gaseous mobile phase (usually helium), called the carrier gas.

A

Gas chromatography

58
Q

What is the use of gas chromatography?

A

Used in analysis urine samples (in conjunction with mass spectrometry) for detection of various metabolites for diagnosis of metabolic disorders, gold standard for detection of illicit drugs.

59
Q

The stationary phase is a nonvolatile liquid bonded to the inside of the column or to a fine solid support

A

Gas-liquid partition chromatography

60
Q

The analyte is adsorbed directly on solid particles of stationary phase.

A

Gas-solid adsorption chromatography

61
Q

Based on the separation of a mixture of compounds as it migrates with the help of a suitable solvent through a thin layer of adsorbent material (stationary phase, most commonly, silica gel) which has been applied to an appropriate support.

A

Thin-layer chromatography

62
Q

What is the application of thin-layer chromatography

A
  1. Determination of the pigments in plants
  2. To detect pesticides or insecticides in food
  3. In forensics to analyze the dye composition of fibers
  4. To identify compounds present in a given substance, among other uses.
63
Q

The system consists of a two immiscible solvent, a nonpolar solvent mixture (mobile phase), water molecules attached to cellulose/ filter paper (stationary phase).

Useful for separating complex mixtures of compounds having similar polarity.

A

Paper chromatography

64
Q

What is the use of paper chromatography?

A

Useful for separating complex mixtures of compounds having similar polarity.

Used primarily in the academic setting to demonstrate the principles of chromatography.

65
Q

What are the physical characteristics that determine the separation of substances in paper chromatography?

A
  1. Rate of diffusion
  2. Solubility of the solute
  3. Nature of the solvent
66
Q

It is the ratio of the distance travelled by the center of the spot to the distance simultaneously travelled by the mobile phase.

A

Rf value

67
Q

Rf formula:

A

Rf = [distance travelled by the solute] x [distance travelled by the solvent]^-1

68
Q

What is the significance of Rf value?

A

The larger anRfof a compound, the larger the distance it travels, hence it has less affinity to the stationary phase.

69
Q

Three test tubes coming from the ion-exchange chromatography containing the 3 amino acids were reserved as for this procedure.
A 20.96 x 57.15cm (8.25 x 22.5in) filter paper is marked accordingly as described from the manual.
The three amino acids were blotted in 3 separates spots in the filter paper using a capillary tube.

A

Ok

70
Q

In the actual performance of this experiment, the amino acids are not yet known at this point.
Instead, another set of 3 known amino acids are usually blotted for reference purposes.
This will allow identification of amino acids by comparing the migration pattern of the spots of unknown samples vs the known references.

A

Ok

71
Q

The blotted papers are now placed inside a chromatography jar containing the solvent mixture.
Solvent was allowed to run down the paper for 13 hour and then allowed to dry.
The amino acid spots at this point are invisible. They have to be identified first by spraying ninhydrin to reveal the location of the amino acid.

A

Ok

72
Q

During the actual performance of this experiment, there is emphasis to avoid touching the chromatography paper with an ungloved hand. Why is this so?

A

Touching the chromatography paper with bare hands would leave traces of amino acids from sweat in the paper which can make interpretation of the chromatogram difficult.

73
Q

Addition of certain salts (usually ammonium sulfate) at appropriate amount to selectively precipitate proteins, which can then be removed by low-speed centrifugation.

A

Salting-out

74
Q

It is a procedure that separates proteins from small solutes by taking advantage of the proteins’ larger size.

A

Dialysis

75
Q

It is a separation of proteins is based on the migration of charged proteins in an electric field.

A

Electrophoresis

76
Q

What are the uses of electrophoresis?

A
  1. Permits quick estimation of the number of different proteins in a mixture or the degree of purity of a particular protein preparation.
  2. It is used to determine crucial properties of a protein such as its isoelectric point and approximate molecular weight.
77
Q

Electrophoresis of proteins is generally carried out in gels made up of the cross-linked polymer __.

A

Polyacrylamide

78
Q

What is the function of polyacrylamide gel in electrophoresis?

A

It acts as a molecular sieve, slowing the migration of proteins approximately in proportion to their charge-to-mass ratio.

79
Q

The migration of the protein in the electrophoresis may also be affected by protein __?

A

Shape

80
Q

It is a procedure used to determine the isoelectric point (pI) of a protein.

A

Isoelectric Focusing

81
Q

In IEF the pH gradient is established by?

A

Allowing a mixture of low molecular weight organic acids and bases to distribute themselves in an electric field generated across the gel.

82
Q

When a protein mixture is applied, each protein migrates until it reaches the pH that matches its pI. Proteins with different isoelectric points are thus distributed differently throughout the gel.

A

True

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