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

Biochem - Protein Characterization and Purification Flashcards

(53 cards)

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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?

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A

at its isoelectric point (pI)

23
Q

2 methods to purify proteins

Study These Flashcards
A

chromatography and electrophoresis

24
Q

What is column chromatography?

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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
Separation by charge process:
- 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
Ionization of amino acids
- 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
Separation by size
- also called size exclusion chromatography - small proteins bind to column - high mw proteins pass more freely so they elute first
28
Separation by affinity
- 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
Hydrophobic interaction chromatography
unique form of chromatography because proteins bind at high salt concentrations and elute at low salt concentrations
30
Electrophoresis separation is based on:
charge, size, and shape of protein
31
Electrophoresis
- gel matrix controls mobility of proteins according to size and shape (mw) - electric field pulls proteins according to their charge (pI)
32
What is SDS (sodium dodecyl sulfate)
- a detergent (in soap)
33
SDS:
- 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
What 2 things make up polyacrylamide gel?
- acrylamide - methylene bisacrylamide
35
What is SDS-Page?
- 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
What is isoelectric focusing (IEF) used for?
To determine the pI of a protein
37
What 2 processes does 2D electrophoresis combine?
- Isoelectric focusing - SDS-Page
38
Define "activity" of an ezyme
has to do with the amount of substrate making product over a period of time
39
What is a way to calculate protein purity?
- specific activity (activity/total protein)
40
What is the units of specific activity?
unit of activity / mg of protein
41
Which protein structure does SDS break down?
- quaternary structure subunits
42
Protein structure characterizations
- primary = amino acid residues - secondary = a helix/beta sheets - tertiary = polypeptide chain - quaternary = 2-4 subunits
43
Spectroscopic detection of aromatic aa's
- 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
Which 2 amino acids are the strongest chromophores?
- tryptophan and tyrosine
45
What 2 things does the detector of a spectrophotometer determine?
- transmitted light and absorption
46
Protein sequencing methods: Edman degradation
- successive rounds of N-terminal modification, cleavage, and identification - can determine the identity of a protein with known sequence
47
Protein sequencing methods: Mass spectrometry
- can identify the mass of a peptide and protein - can also determine post-translational modifications
48
3 most used chemical cleavage agents:
- cyanogen bromide - chymotrypsin - trypsin
49
Mass spectrometry
- used to determine ionized forms of molecules in the gas phase - applies a positive charge on ions - 2 types: MALDI and ESI
50
Is mass spec used for qualitatively or quantitative analysis?
both
51
Mass spec overview
- sample - ion source (become +) - mass analyzer (in vacuum) - detector
52
In mass spec, what does the tallest peak represent?
base peak of the molecular ion
53
In mass spec, what does the smallest peak all the way to the right represent?
molecular ion isotope peak

Integrated Exam 3/9 (8 decks)

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