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Integrated Exam 3/9 > Biochem - Protein Characterization and Purification > Flashcards

Biochem - Protein Characterization and Purification Flashcards

1
Q

What type of protein has covalently linked a-amino acids?

A

polypeptides

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2
Q

What is a cofactor?

A

a functional non-amino acid that is bound to proteins

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3
Q

Are cofactors organic or inorganic?

A

they can be either

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4
Q

What is an example of a cofactor?

A

inorganic: metal ions
organic: vitamins

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5
Q

What are coenzymes?

A

organic cofactors

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6
Q

What is an example of a coenzyme?

A
  • NAD+ in lactase dehydrogenase
  • vitamins
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7
Q

What are prosthetic groups?

A

covalently attached cofactors

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8
Q

What is an example of a prosthetic groups?

A

heme in myoglobin

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9
Q

Classes of conjugated proteins

A
  • lipoproteins
  • glycoproteins
  • phosphoproteins
  • hemoproteins
  • flavoproteins
  • metalloproteins
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10
Q

1st step in protein purification

A

isolate and purify the protein

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11
Q

What is Isopycnic (sucrose-density) centrifugation?

A
  • used for fractionating of DNA, RNA, and proteins
  • a mixture of different sized macromolecules is layered on the surface of a gradient whose density increases from top to bottom
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12
Q

What is differential centrifugation?

A

a separation method where components of a cell are separated based on their density in a centrifuge according to the centrifugal force they experience (low speed vs medium speed vs high speed)

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13
Q

What is the result of differential centrifugation?

A

a pellet consisting of ribosomes and large macromolecules

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14
Q

What 2 differences do separation techniques rely on?

A

physical and chemical properties

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15
Q

Examples of protein properties

A
  • solubility
  • charge
  • size
  • affinity for a ligand
  • hydrophobicity
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16
Q

What is meant by salting out?

A

proteins are less soluble at high salt concentrations

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17
Q

How are salt concentration and solubility related?

A

high salt con = low solubility

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18
Q

What is salting out used to determine?

A
  • different proteins precipitate at different salt concentrations
  • salting out can be used to fractionate proteins
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19
Q

What can be used to remove salt?

A

Dialysis

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20
Q

What interactions does “salting in” supress?

A
  • suppresses the electrostatic protein-protein interactions
  • goal of salting in is to INCREASE solubility of proteins
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21
Q

What is the isoelectric point of a protein?

A

the pH at which the protein has no net charge

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22
Q

When is a protein least soluble?

A

at its isoelectric point (pI)

23
Q

2 methods to purify proteins

A

chromatography and electrophoresis

24
Q

What is column chromatography?

A
  • a technique used to purify compounds depending on their polarity or hydrophobicity
  • nonpolar compounds come out first
  • polar compounds elute last
  • 2 phases: mobile and stationary
25
Q

Separation by charge process:

A
  • protein mixture is added to column with cation exchanger
  • proteins move through column at rates determined by their net charge at the pH
  • neg net charge proteins move faster
  • neg net charge elute first
26
Q

Ionization of amino acids

A
  • acidic pH (lower than pI) = carboxyl is neutral and amino group is protonated (aa is in cationic form)
  • neutral pH (net charge 0) = carboxyl is deprotonated and amino group is protonated = zwitterion
  • alkaline pH (higher than pI) = carboxyl is deprotonated and amino groups is neutral (aa is in anionic form)
27
Q

Separation by size

A
  • also called size exclusion chromatography
  • small proteins bind to column
  • high mw proteins pass more freely so they elute first
28
Q

Separation by affinity

A
  • protein mixture is added to column that has polymer bound ligand for a specific protein
  • protein of interest will bind
  • unwanted proteins will wash through the column
  • another ligand is then added in order to strip the wanted ligand from the column
29
Q

Hydrophobic interaction chromatography

A

unique form of chromatography because proteins bind at high salt concentrations and elute at low salt concentrations

30
Q

Electrophoresis separation is based on:

A

charge, size, and shape of protein

31
Q

Electrophoresis

A
  • gel matrix controls mobility of proteins according to size and shape (mw)
  • electric field pulls proteins according to their charge (pI)
32
Q

What is SDS (sodium dodecyl sulfate)

A
  • a detergent (in soap)
33
Q

SDS:

A
  • SDS micelles bind and unfold proteins
  • gives all proteins an uniformly neg charge
  • rate of movement only depends on size (small proteins will move faster)
34
Q

What 2 things make up polyacrylamide gel?

A
  • acrylamide
  • methylene bisacrylamide
35
Q

What is SDS-Page?

A
  • SDS-PAGE is used for protein separation by MASS (size)
  • SDS give proteins an uniformly neg charge
  • molecules are going from anode to cathode
36
Q

What is isoelectric focusing (IEF) used for?

A

To determine the pI of a protein

37
Q

What 2 processes does 2D electrophoresis combine?

A
  • Isoelectric focusing
  • SDS-Page
38
Q

Define “activity” of an ezyme

A

has to do with the amount of substrate making product over a period of time

39
Q

What is a way to calculate protein purity?

A
  • specific activity (activity/total protein)
40
Q

What is the units of specific activity?

A

unit of activity / mg of protein

41
Q

Which protein structure does SDS break down?

A
  • quaternary structure subunits
42
Q

Protein structure characterizations

A
  • primary = amino acid residues
  • secondary = a helix/beta sheets
  • tertiary = polypeptide chain
  • quaternary = 2-4 subunits
43
Q

Spectroscopic detection of aromatic aa’s

A
  • aromatic aa’s absorb light in the UV region
  • protein absorbance max is 275-280 nm
  • concentration is determined by UV visible spec using Beers law
44
Q

Which 2 amino acids are the strongest chromophores?

A
  • tryptophan and tyrosine
45
Q

What 2 things does the detector of a spectrophotometer determine?

A
  • transmitted light and absorption
46
Q

Protein sequencing methods: Edman degradation

A
  • successive rounds of N-terminal modification, cleavage, and identification
  • can determine the identity of a protein with known sequence
47
Q

Protein sequencing methods: Mass spectrometry

A
  • can identify the mass of a peptide and protein
  • can also determine post-translational modifications
48
Q

3 most used chemical cleavage agents:

A
  • cyanogen bromide
  • chymotrypsin
  • trypsin
49
Q

Mass spectrometry

A
  • used to determine ionized forms of molecules in the gas phase
  • applies a positive charge on ions
  • 2 types: MALDI and ESI
50
Q

Is mass spec used for qualitatively or quantitative analysis?

A

both

51
Q

Mass spec overview

A
  • sample
  • ion source (become +)
  • mass analyzer (in vacuum)
  • detector
52
Q

In mass spec, what does the tallest peak represent?

A

base peak of the molecular ion

53
Q

In mass spec, what does the smallest peak all the way to the right represent?

A

molecular ion isotope peak

Integrated Exam 3/9 (8 decks)

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