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RED - Chemistry > Physical Chemistry of Proteins/Analytical Methods > Flashcards

Physical Chemistry of Proteins/Analytical Methods Flashcards

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1
Q

How do histidine side chains often fulfill important functions?

A

Its pKa is lose to physiological pH (6 vs. 7.4); where acid/base catalysis occurs in some proteases

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2
Q

How does the ionisation state of the histidine side chain change with pH?

A
  • Imidazole ring is mostly protonated below pH 6; carrying a positive charge equally distributed between both nitrogens.
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3
Q

What do posttranslational modifications of proteins achieve?

A

It can influence the physicochemical properties of proteins, resulting in potential changes in solubility, stability, bonding etc.

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4
Q

Which hydroxyl groups are commonly phosphorylated in posttranslational modifications and what can this achieve?

A
  • Ser, Thr, Tyr
  • Phosphorylation gives -ve charge and increases bulk; encouraging protein-proteins interaction
  • E.g. Tyr phosphorylation in tyrosine kinase receptors; key to signalling pathways in the cell
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5
Q

What does glycosylation entail and how does it affect the physio-chemical properties of proteins?

A
  • Attachment of sugar moieties to Ser, Thr or Asn residues

- Can alter solubility ( -OH group makes protein more soluble/stable)

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6
Q

What does hydroxylation entail (and to which AAs) and how does it influence the physio-chemical properties proteins?

A
  • Addition of hydroxyl (OH) group
  • To Pro or Lys residues
  • Can alter H-bonding
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7
Q

What does methylation entail and how does it influence the physio-chemical properties or proteins?

A
  • Addition of methyl groups (CH3) to N or O atoms of AA side chains
  • Added hydrophobic (non-polar) group
  • Thus sterically bigger
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8
Q

What does disulfide bond formation entail and what effect does it have on the physio-chemical properties of proteins?

A
  • Covalent bond between two Cys AAs (thiol groups/sulfur)

- Renders protein more stable due to additional covalent linkage

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9
Q

If a protein has many basic side chains, what charge will it possess at physiological pH?

A
  • Protein is positively charged

- Abundance of basic side chains = easily protonated = H+

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10
Q

If a protein has mainly acidic side chains, what charge will it possess at physiological pH?

A
  • Protein is negatively charged

- Easily deprotonated = COO-

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11
Q

What factors influence a protein’s state of ionisation?

A
  • Amino acids

- pH of the solution environment

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12
Q

What is the isoelectric point (IEP)?

A

The pH at which a particular molecule molecule or surface carries no net electrical charge; does not migrate in an electric field.

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13
Q

What does a protein being at its IEP influence, and what’s the common range for protein IEP?

A
  • Protein is at its least soluble
  • But this also means it’s at its most permeable
  • Occurs for most proteins at pH 5.5 - 8
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14
Q

How is the influence of IEP on solubility be exploited in an insulin glargine (long-acting) formulation?

A
  • Insulin glargine contains two extra Arg residues at the end of the B-chain
  • Arg residues are basic
  • Thus raising the IEP and altering its solubility; it is more soluble in the acid conditions used in the formulation (more protonation) but less soluble upon injection (physiological pH)
  • Insulin precipitates out and slowly dissolves in blood (due to raised IEP) giving long-lasting action
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15
Q

What can the separation of different proteins be based upon?

A
  • Differences in charge
  • Differences in hydrophobicity
  • Differences in solubility
  • Differences in size
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16
Q

What is the principle of gel electrophoresis?

A
  • Molecules separated according to their size and charge (size-to-charge ratio)
  • Proteins and nucleic acids electrophoresed within a matrix or “gel”
  • Charged molcules migrate toward either postive or negative pole according to charge in an electric field
17
Q

What are the different forces of attraction and retardation at play in gel electrophoresis?

A

Attraction:

  • Size of charge
  • Size of electric field

Retardation:

  • Friction (of the matrix)
  • Repulsion in medium
18
Q

What factors give a protein high mobility in gel electrophoresis?

A
  • Small

- Highly charged

19
Q

What factors give a protein low mobility in gel electrophoresis?

A
  • Large

- Minimally charged

20
Q

What are the requirements for the analytes in electrophoresis and why?

A
  • Must be charged (or have a charge induced)
  • Usually contain acidic or basic functional groups
  • Can’t be at IEP; ionisation dependent on pKa of functional group and pH of electrolyte
21
Q

What are the benefits of using Native Polyacrylamide Gel Electrophoresis (PAGE) over other electrophoresis?

A
  • Native structure of protein maintained during electrophoresis (3D structure not disrupted)
  • Separation according to SIZE and CHARGE
22
Q

How dose PAGE achieve keeping native protein structure?

A
  • Protein isn’t denatured; acrylamide gel is a size-selective sieve during separation
  • Smaller molecules travel more rapidly than larger proteins through the gel in response to an electric field
23
Q

What does SDS-polyacrylamide gel electrophoresis entail?

A
  • Separation of proteins according to SIZE only
  • Native (3D) structure of protein is not maintained; proteins are denatured by heat and the addition of the detergent SDS prior to electrophoresis
24
Q

How is it ensured that proteins are only separated by size in SDS-polyacylamide gel electrophoresis?

A
  • Via the addition of sodium dodecyl sulfate
  • A detergent; emulsifies protein and gives a net negative charge, with different proteins in the same SDS solution given approximately the same charge:mass ratio; predominantly migrating on size
  • Hydrophobic tail interacts with oil/lipid membranes, negative hydrophilic head sticks out; making it water soluble too
25
Q

What are the typical detection methods for proteins post-electrophoresis?

A
  • Coomassie Brilliant blue dye staining (Bradford assay)

- Western blot (protein immunoblot); transfer of gel contents onto a membrane and detection via labelled antibodies

26
Q

What are the advantages of UV absorption (spectrophotometry)?

A
  • No additional reagents or incubations required
  • No protein standard needs to be prepared
  • Assay does not consume protein
  • Relationship of absorbance to protein concentration is linear
27
Q

What are the disadvantagse of UV absorption (spectrophotometry)?

A
  • Any non-protein component of the solution that absorbs UV light will interfere with assay
28
Q

Why are there maxima at 280 nm and 200 nm with protein absorbance UV?

A

280nm: amino acids with aromatic rings (Trp and Tyr)
200nm: peptide bonds

29
Q

What other factors can affect the absorbance spectrum and why?

A
  • Secondary, tertiary and quaternary structure all affect absorbance
  • Thus factors such as pH, ionic strength etc. affect the spectrum
30
Q

What colorimetric methods for measuring protein concentration are available and what can be generated as a result?

A
  • Bradford assay (dye based)
  • BCA Protein Assay (copper based)

A standard curve with samples of known protein concentrations can be created, and thus the concentration of the unknown protein is determined from the curve.

31
Q

What does the Bradford assay entail?

A
  • Colorimetric method for measuring protein concentration
  • Dye based
  • Coomassie Brilliant Blue dye binds to proteins in acidic solution (via electrostatic and van der Waals forces)
  • Results in shift of absorption maxima of dye from 465 to 595 nm
32
Q

What does the BCA Protein Assay entail?

A
  • Colorimetric method for measuring protein concentration
  • Copper based
  • Reduction of Cu2+ to Cu+ by protein in an alkaline medium with subsequent colorimetric detection of Cu+ cation by bicinchoninic acid
  • Intense purple-coloured reaction product results from chelation of two molecules of BCA with one Cu+ ion
  • BCA/Cu+ complex exhibits strong linear absorbance at 562nm with increasing protein concentrations
33
Q

What is the advantage of using the BCA Protein Assay to the Bradford assay?

A

Peptide backbone (that reduces Cu2+ in alkaline) also contributes to colour formation, helping to minimise variability caused by protein compositional differences.

RED - Chemistry (3 decks)

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