Biochem - Protein Characterization and Purification Flashcards
What type of protein has covalently linked a-amino acids?
polypeptides
What is a cofactor?
a functional non-amino acid that is bound to proteins
Are cofactors organic or inorganic?
they can be either
What is an example of a cofactor?
inorganic: metal ions
organic: vitamins
What are coenzymes?
organic cofactors
What is an example of a coenzyme?
- NAD+ in lactase dehydrogenase
- vitamins
What are prosthetic groups?
covalently attached cofactors
What is an example of a prosthetic groups?
heme in myoglobin
Classes of conjugated proteins
- lipoproteins
- glycoproteins
- phosphoproteins
- hemoproteins
- flavoproteins
- metalloproteins
1st step in protein purification
isolate and purify the protein
What is Isopycnic (sucrose-density) centrifugation?
- 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
What is differential centrifugation?
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)
What is the result of differential centrifugation?
a pellet consisting of ribosomes and large macromolecules
What 2 differences do separation techniques rely on?
physical and chemical properties
Examples of protein properties
- solubility
- charge
- size
- affinity for a ligand
- hydrophobicity
What is meant by salting out?
proteins are less soluble at high salt concentrations
How are salt concentration and solubility related?
high salt con = low solubility
What is salting out used to determine?
- different proteins precipitate at different salt concentrations
- salting out can be used to fractionate proteins
What can be used to remove salt?
Dialysis
What interactions does “salting in” supress?
- suppresses the electrostatic protein-protein interactions
- goal of salting in is to INCREASE solubility of proteins
What is the isoelectric point of a protein?
the pH at which the protein has no net charge
When is a protein least soluble?
at its isoelectric point (pI)
2 methods to purify proteins
chromatography and electrophoresis
What is column chromatography?
- 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
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
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)
Separation by size
- also called size exclusion chromatography
- small proteins bind to column
- high mw proteins pass more freely so they elute first
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
Hydrophobic interaction chromatography
unique form of chromatography because proteins bind at high salt concentrations and elute at low salt concentrations
Electrophoresis separation is based on:
charge, size, and shape of protein
Electrophoresis
- gel matrix controls mobility of proteins according to size and shape (mw)
- electric field pulls proteins according to their charge (pI)
What is SDS (sodium dodecyl sulfate)
- a detergent (in soap)
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)
What 2 things make up polyacrylamide gel?
- acrylamide
- methylene bisacrylamide
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
What is isoelectric focusing (IEF) used for?
To determine the pI of a protein
What 2 processes does 2D electrophoresis combine?
- Isoelectric focusing
- SDS-Page
Define “activity” of an ezyme
has to do with the amount of substrate making product over a period of time
What is a way to calculate protein purity?
- specific activity (activity/total protein)
What is the units of specific activity?
unit of activity / mg of protein
Which protein structure does SDS break down?
- quaternary structure subunits
Protein structure characterizations
- primary = amino acid residues
- secondary = a helix/beta sheets
- tertiary = polypeptide chain
- quaternary = 2-4 subunits
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
Which 2 amino acids are the strongest chromophores?
- tryptophan and tyrosine
What 2 things does the detector of a spectrophotometer determine?
- transmitted light and absorption
Protein sequencing methods: Edman degradation
- successive rounds of N-terminal modification, cleavage, and identification
- can determine the identity of a protein with known sequence
Protein sequencing methods: Mass spectrometry
- can identify the mass of a peptide and protein
- can also determine post-translational modifications
3 most used chemical cleavage agents:
- cyanogen bromide
- chymotrypsin
- trypsin
Mass spectrometry
- used to determine ionized forms of molecules in the gas phase
- applies a positive charge on ions
- 2 types: MALDI and ESI
Is mass spec used for qualitatively or quantitative analysis?
both
Mass spec overview
- sample
- ion source (become +)
- mass analyzer (in vacuum)
- detector
In mass spec, what does the tallest peak represent?
base peak of the molecular ion
In mass spec, what does the smallest peak all the way to the right represent?
molecular ion isotope peak
