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ENDOCRINE > C. PROTEIN CHEMISTRY 3 > Flashcards

C. PROTEIN CHEMISTRY 3 Flashcards

1
Q

post-translational modifications of proteins

A
  • phosphorylation of hydroxyl groups Ser, Thr or Tyr
  • can or can’t make interactions due to added charge
  • ie:
    1. Tyr phosphorylation of receptor
    Tyrosine kinase receptors
    Phosphorylations are key to signalling pathways in the cell
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2
Q

what are the 4 types of post translational modifications

A
  1. Glycosylation
    - Attachment of sugar
    moieties to Ser, Thr or Asn residues, can alter solubility (more stable)
  2. Hydroxylation
    - Hydroxyl group (OH) added
    to Pro or Lys residues, can alter hydrogen
    bonding
  3. Methylation: Methyl groups can be added
    to nitrogen or oxygen atoms of amino acid side chains: added hydrophobic group
  4. Disulfide bond formation between two cysteines, typically renders proteins more stable: additional covalent linkage
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3
Q

if a protein has many basic side chains, what is the overall charge at physiological pH

A

net +ve charge

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4
Q

if a protein has many acidic side chains, what is the overall charge at physiological pH

A

net -ve charge

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5
Q

if a protein has acidic and basic side chains, what is the overall charge at physiological pH

A

whichever one is more frequent

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6
Q

what determines the protein’s state of ionisation

A
  • amino acids
  • pH of the solution environment
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7
Q

what is the isoelectric point (pI)

A

pH at which the molecule or surface carries no net electrical charge

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8
Q

what are the 2 consequences of the pI

A
  1. doesn’t migrate in an electric field
  2. protein is least soluble

normally pH range of 5.5-8

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9
Q

what is the influence of pI on solubility

A
  • Insulin glargine is a long-acting insulin that contains two extra arginine residues (basic) at the end of the B chain
  • increases isoelectric point of insulin (normally ~5.4) to 6 ish and alters the solubility, more soluble in the acidic
    conditions (pH 4) used in the formulation, but
    less soluble upon injection (encounters pH 7.4)
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10
Q

what is separation of different proteins based on

A
  • charge
  • hydrophobicity
  • solubility
  • size
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11
Q

what is gel electrophoresis

A
  • for separation and analysis of macromolecules (DNA and proteins)
  • separated according to their size and charge
  • electrophoresed within a matrix or “gel”
  • in an electric field charged molecules migrate toward either the positive or
    negative pole according to their charge
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12
Q

where do anions move towards

A

the anode (+ve)

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13
Q

where do cations move towards

A

the cathode (-ve)

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14
Q

what are the forces of attraction

A
  • size of charge
  • size of electric field
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15
Q

forces of retardation

A
  • friction
  • repulsion in medium
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16
Q

what type of species have high mobility

A

small, highly charged species

17
Q

what type of species have low mobility

A

large, minimally charged species

18
Q

how does the structure the analyte affect migration

A
  • analytes must be charged/have a charge induced
  • contain acidic or basic functional groups
  • ionisation dependent on pKa of functional groups and pH of electrolyte
  • pH>pKa = deprotonated
19
Q

what is native polyacrylamide gel electrophoresis (PAGE)

A
  • ‘native’ structure of protein is maintained during electrophoresis
  • separation according to size and charge
20
Q

how does size affect separation

A
  • acrylamide gels serve as a size-selective sieve during separation
  • as proteins move through a gel in response to an electric field, the smaller molecules travel more rapidly
21
Q

how does charge affect separation

A
  • highly negatively charged molecules will migrate faster than less negatively charged molecules towards the anode
  • these two effects in combination can mean that a highly negatively charged larger molecule can migrate faster than a less negatively charged smaller molecule
22
Q

applications of native PAGE electrophoresis

A
  • assess quaternary structure (oligomerisation state) of a protein
  • homogeneity of a pure protein sample

ie - insulin, might see a band for dimer and a band for the hexamer

23
Q

what is SDS-polyacrylamide gel electrophoresis

A
  • ‘native’ structure of proteins is NOT maintained during electrophoresis: proteins are denatured
  • separation according to SIZE
  • proteins are denatured by heat and the addition of the detergent SDS (Sodium dodecyl sulfate) prior to electrophoresis
24
Q

sodium dodecyl sulfate trick

A
  • surfactant = polar to aq sol and non-polar to oil on top
  • emulsification by the detergent (SDS) gives proteins a net negative charge
  • different proteins in the same SDS solution are imparted with approximately the same charge to mass ratio, so will predominantly migrate according to size alone
25
Q

what are the 2 ways to detect proteins post-electrophoresis

A
  1. Coomassie Brilliant blue dye staining
    - dye needs to have conjugated double bonds to absorb visible light and hence show colour
  2. Western blot (protein immunoblot)
    - transfer of gel contents onto a membrane and detection via labelled antibodies
26
Q

how to measure protein concentrations

A

UV absorption
- a dissolved substance will absorb light of specific wavelengths characteristic of that substance
- Extinction coefficient/Lambert Beer Law

27
Q

advantages of UV absorption

A
  • no additional reagents or incubations are required
  • no protein standard need to be prepared
  • the assay does not consume the protein
  • the relationship of absorbance to protein concentration is linear
28
Q

disadvantages of UV absorption

A
  • any non-protein component in the solution that absorbs ultraviolet light will interfere with the assay
29
Q

what are the absorbance maximums

A
  • proteins in solution absorb ultraviolet light with absorbance maxima at 280 nm and 200 nm
  • 280 nm: mainly due to amino acids with aromatic rings
  • 200 nm: mainly due to peptide bonds
  • secondary, tertiary, and quaternary structure all affect absorbance, therefore factors such as pH, ionic
    strength, etc. can alter the absorbance spectrum
30
Q

what are the 2 colorimetric methods for measuring protein concentrations

A
  1. Bradford assay: dye based
  2. BCA protein assay: copper based

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

31
Q

Bradford assay: dye based

A
  • Coomassie Brilliant Blue dye binds to proteins in acidic solution (via electrostatic and van der Waals bonds) resulting in a shift of the absorption maximum of the dye from 465 to 595 nm
32
Q

BCA protein assay: copper based

A
  • principle: reduction of Cu2+ to Cu1+ by protein in an alkaline medium with colorimetric detection of the cuprous cation (Cu1+) by bicinchoninic acid (BCA)
  • intense purple-colored reaction product results from the chelation of two molecules of BCA with one cuprous ion
  • the BCA/copper complex exhibits a strong linear absorbance at 562 nm with increasing protein concentrations
33
Q

advantage of BCA Protein Assay: copper based

A
  • unlike the Coomassie dye-binding methods, the peptide backbone also contributes to colour formation, helping to minimize variability caused by protein compositional difference

ENDOCRINE (17 decks)

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