Shared Flashcard Set

Details

BCHM315 - Test #1
N/A
64
Biochemistry
Undergraduate 3
10/10/2011

Additional Biochemistry Flashcards

 


 

Cards

Term
Main properties of H2O
Definition
Polarity (ability to form H-bonds in solution)
Ability to ionize (act as weak acid/weak base in solution)
Term
H-bonds are what type of interactions?
Definition
Electrostatic
Term
Typical length of an O-H bond? Length of an H-bond in water?
Definition
O-H = 1 Angstrom
H-bond = 1.8 Angstroms
Term
H-bond is....?
Definition
Directional -> O---H-O bond is linear (forms a straight line)
Term
In ice, one molecule of water forms how many neighboring H-bonds?
Definition
Forms H-bonds with 4 neighbors (maximum)
Term
Average lifetime of 1 H-bond?
Definition
10^-12 seconds = 1 picosecond
Term
In liquid water, how many neighboring H-bonds & why?
Definition
3.4 bonds w/ neighbors; due to there being increased entropy in this state (less order)
Term
Thermodynamic reasoning for the dissolution of glucose...
Definition
It is a polar solute, so it can readily form H-bonds with water; as the solid disperses in the water, the entropy of the system increases (S>0); entropic effect of less of order of solute is main reason
Term
Thermodynamic reasoning for dissolution of salt in water...
Definition
Normally as a solid, see Na & Cl ions in crystal lattice structure; as it enters water, see electrostatic attraction of dipole in water molecules to the ions (6 moc H2O/ion) - causes entropy of the whole system to increase (freeing of ions from lattice)
Term
Main interaction for NON-polar solutes in water?
Definition
van der Waal's interactions - transient dipoles based on varying electron densities of atoms; NOT TRUE ELECTROSTATIC INTERACTIONS
Term
Non-polar solute aggregation in water is known as...
Definition
The Hydrophobic Effect; aggregation is mainly an ENTROPIC EFFECT (solutes aggregate to minimize SA exposed to water, so water becomes less ordered)
Term
What is the primary contributor to protein conformational changes?
Definition
The hydrophobic effect
Term
Concentration of pure water
Definition
55.5 M
Term
If [A]/[HA] = 0.1
Definition
pH = pKa - 1 (90% HA, 10% A)
Term
If [A]/[HA] = 1
Definition
pH = pKa (50% HA, 50% A)
Term
If [A]/[HA] = 10
Definition
pH = pKa + 1 (10% HA, 90% A)
Term
All but one aa is chiral, which is not?
Definition
Glycine
Term
Which amino acid is NOT S (has R chirality)?
Definition
Cysteine because of SH group
Term
Isoelectric Point
Definition
The point at which a molecule has no net charge
Term
Residue Mass = ?
Definition
R Mass = Amino Acid Mass - 18 (MW of water)

In a polypeptide, the N and C-terminal amino acids will be represented by amino acid MW and the internal aa's will be represented by residue masses
Term
What is isoelectric focusing used for?
Definition
To separate different proteins based on different pI values; to determine the unknown pI value of a protein
Term
SDS-PAGE is what method of protein purification?
Definition
Semi-quantitative method; determine the relative amounts of proteins in a sample (mole ratios)
Term
What property does SDS-PAGE use to separate proteins?
Definition
Separates proteins based on differences in MW (all proteins w/ SDS have same charge to mass ratio and roughly the same shape)
Term
What is proportional to the amount of dye that binds to a protein?
Definition
The protein's mass; heavier proteins bind more dye
Term
What interactions typically hold oligomeric proteins together? How can we separate the individual protomers?
Definition
Hydrophobic interactions
Can use SDS to separate protomers - binds to hydrophobic surfaces which causes protomers to separate
Term
What conditions are S-S bonds formed/broken in?
Definition
Formed = OXIDIZING
Broken = REDUCING
Term
2D Gel Electrophoresis...
Definition
Combination of IEF and SDS PAGE; first use IEF to separate proteins based on pI, then SDS PAGE to separate based on MW
Term
Where do aromatic aa side chains absorb mainly at?
Definition
Wavelength = 280 nm
Term
What are limitations of using A280 (absorbance) to measure the purity and amounts of a protein sample?
Definition
Not very sensitive (only to 50 micrograms/mL)
Can only use on random mixes; cannot be done on pure samples
Term
If you have a pure sample of known F/Y/W composition, how can you calculate the concentration?
Definition
Use the known relative amounts to determine the extinction coefficient -> then rearrange Beer's Law formula to calculate the concentration of protein
Term
Can SDS break S-S bonds alone?
Definition
NO. Need to add a reducing agent to break bonds
Term
What interactions cause dye molecules to associate with proteins?
Definition
Hydrophobic effect
Term
Enzyme Unit
Definition
1 unit = amount of protein it takes to convert 1 micromole of substrate in 1 minute at 25 degrees C
Term
Specific Activity
Definition
Measure of purity; # of units/total protein (mg)
Term
What is typically the 1st step in a purification process?
Definition
Separation of proteins based on solubility differences
Term
Solubility at LOW [salt]...
Definition
Charges on different side-chains cause electrostatic interactions between proteins and they aggregate and precipitate out of solution (LOW solubility); little hydrophobic effects because [salt] is low
Term
Solubility as [salt] increases...
Definition
As the [salt] increases, the salt ions coat the proteins in "compensating charges" to reduce interactions (shields proteins) - less aggregation (HIGH solubility)
Term
Solubility at HIGH [salt]...
Definition
At very high [salt] there are so many ions that they shield water molecules as well, interrupting with the H-bonding and other intermolecular interactions between the water and protein; less H2O to solvate proteins
Hydrophobic surfaces of proteins aggregate together to cause ppt out of solution (LOW solubility) - aggregation due to hydrophobic effects
Term
If pH < pI
Definition
Then the protein is NET POSITIVE - charges repel each other (HIGH solubility)
Term
If pH = pI
Definition
Proteins aggregate due to hydrophobic effects because they are neutral (LOW solubility)
Term
If pH > pI
Definition
Proteins are NET NEGATIVE; charges repel and proteins remain in solution (HIGH solubility)
Term
Typical column molecule in CATION EXCHANGE?
Definition
Carboxymethyl cellulose (-CH2-COO)
Term
Why is the hydrophobic effect more prevalent at high [salt]?
Definition
It is due to an excess of ions in the solution which tie up more water molecules; this prevents them from solvating the proteins which allows for more hydrophobic binding (higher affinity to binding)
Term
Void Volume
Definition
Eluted volume of proteins TOO LARGE for the pores in size exclusion chromotography; = volume outside of the beads in the column
Term
Total Volume
Definition
Eluted volume of proteins which traveled through the beads in size exclusion chromotography; = void volume + volume of pores of beads
Term
What is the ratio of elution volume/void volume dependent on?
Definition
The shape of the protein itself; rod-shaped proteins of identical MW to spherical proteins will elute FASTER in size exclusion columns because they behave as larger proteins (length of rod = diameter of "larger protein")
Term
Edman Reagent
Definition
Phenylisothiocyanate (PITC); benzene ring-N=C=S

Reacts with ALL free amino groups (including those at N-terminus and on Lys)
Term
What can be used to recognize the PTHaa removed by Edman degradation?
Definition
HPLC (combination of cation exchange and hydrophobic chromotography)
Term
What conditions is Edman degradation run under?
Definition
ALKALINE - need alkaline conditions to make the amino group a good nucleophile
Term
Limitations of Edman Degradation
Definition
PITC reacts twice with Lys side chains; however, only cleaves the peptide bond
Limitation of less than 100 aa's
Often there is NO free N-terminus
Term
Cleavage Sites of Enzymes
Definition
Trypsin - after K, R
Chymotrypsin - after F, Y, W
Elastase - after G, A, S, V
V8 Protease - after D, E
Asp-N - before D, E

****Serine proteases do not cleave before (on N-terminus side of) Pro
Term
Problems with A.A. Composition sequencing?
Definition
Destroys all Trp
Hydrolyzes all amide bonds present -> converts all Asn to Asp and all Gln to Glu
Term
Qualifications for a "similar" sequence?
Definition
Greater than/equal to 25% sequence identity
Term
Divergent evolution will produce which form of homologue?
Definition
Paralogue - two similar proteins with usually different functions within the same organism
Term
Orthologues
Definition
2 similar proteins in different organisms with the same function
Term
Theta (for affinity)
Definition
Fraction of bound receptors ([PL]/[P] + [PL])
Term
How does a molecule of dye bind to a protein?
Definition
Binds hydrophobically (hydrophobic effect); will bind independent of the aa composition
Term
To use standards to determine MW, need 2 things:
Definition
Need void volume < elution volume
Plot graph of Ve/Vo vs. logMW
Term
How to elute affinity chromotography column?
Definition
Add ligand in solution in free form (attached to nothing); binds to protein in mobile phase and causes it to bind less w/ the column, elutes
Term
When using a His-tag, what is used to elute the column?
Definition
Add in free IMIDAZOLE; mirrors the His side chain so that it competes for binding with the metals on the column (Co and Ni)
Term
What is used to recognize PTHaa's?
Definition
Characteristic elution times in a column; NOT the wavelength of absorption
Term
What is the byproduct of cleavage w/ CNBr?
Definition
Homoserine lactone where the Met residue was located
Term
What molecule can be used to block S-S bonds from forming?
Definition
Iodoacetate
Term
Problems w/ Mass Spec
Definition
Leu and Ile cannot be distinguished; both have the same molecular weight
Supporting users have an ad free experience!